Azocane and azonane derivatives and methods of treating hepatitis b infections

ABSTRACT

Provided herein are compounds useful for the treatment of HBV infection in a subject in need thereof, pharmaceutical compositions thereof, and methods of inhibiting, suppressing, or preventing HBV infection in the subject.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/135,243, filed on Mar. 19, 2015. The entire content of thisapplication is herein incorporated by reference.

BACKGROUND

Chronic hepatitis B virus (HBV) infection is a significant global healthproblem, affecting over 5% of the world population (over 350 millionpeople worldwide and 1.25 million individuals in the U.S.).

Despite the availability of a prophylactic HBV vaccine, the burden ofchronic HBV infection continues to be a significant unmet worldwidemedical problem, due to suboptimal treatment options and sustained ratesof new infections in most parts of the developing world. Currenttreatments do not provide a cure and are limited to only two classes ofagents (interferon and nucleoside analogues/inhibitors of the viralpolymerase); drug resistance, low efficacy, and tolerability issueslimit their impact. The low cure rates of HBV are attributed, at leastin part, to the presence and persistence of covalently closed circularDNA (cccDNA) in the nucleus of infected hepatocytes. However, persistentsuppression of HBV DNA slows liver disease progression and helps preventhepatocellular carcinoma. Current therapy goals for HBV-infectedpatients are directed to reducing serum HBV DNA to low or undetectablelevels, and reducing or preventing the development of cirrhosis andhepatocellular carcinoma.

There is a need in the art for therapeutic agents that treat, ameliorateor prevent HBV infection. Administration of these therapeutic agents toan HBV infected patient, either as monotherapy or in combination withother HBV treatments or ancillary treatments, will lead to significantlyimproved prognosis, diminished progression of the disease, and enhancedseroconversion rates.

SUMMARY OF THE INVENTION

Provided herein are compounds useful for the treatment of HBV infectionin a subject in need thereof.

In one aspect, provided herein are compounds having the Formula I:

or a pharmaceutically acceptable salt thereof, wherein

Cy is

In another aspect, provided herein is a composition comprising acompound of Formula I, or a pharmaceutically acceptable salt thereof.

In an embodiment, the composition is a pharmaceutical composition andfurther comprises at least one pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound ofFormula I.

In yet another aspect, provided herein is a method of reducing the viralload associated with an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of Formula I.

In still another aspect, provided herein is a method of reducingreoccurrence of an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound according to of Formula I.

Also provided herein is a method of reducing an adverse physiologicalimpact of an HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of Formula I.

In another aspect, provided herein is a method of inducing remission ofhepatic injury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of Formula I.

In yet another aspect, provided herein is a method of reducing thephysiological impact of long-term antiviral therapy for HBV infection inan individual in need thereof, comprising administering to theindividual a therapeutically effective amount of a compound of FormulaI.

In still another aspect, provided herein is a method of prophylacticallytreating an HBV infection in an individual in need thereof, wherein theindividual is afflicted with a latent HBV infection, comprisingadministering to the individual a therapeutically effective amount of acompound of Formula I.

Any of the above methods may further comprise administration to theindividual at least one additional therapeutic agent. In an embodiment,the additional therapeutic agent is selected from the group consistingof a HBV polymerase inhibitor, immunomodulatory agents, pegylatedinterferon, viral entry inhibitor, viral maturation inhibitor, capsidassembly modulator, reverse transcriptase inhibitor, a cyclophilin/TNFinhibitor, a TLR-agonist, and an HBV vaccine, and a combination thereof.

In another embodiment, the therapeutic agent is a reverse transcriptaseinhibitor, and is at least one of Zidovudine, Didanosine, Zalcitabine,2′,3′-dideoxyadenosine, Stavudine, Lamivudine, Abacavir, Emtricitabine,Entecavir, Apricitabine, Atevirapine, ribavirin, acyclovir, famciclovir,valacyclovir, ganciclovir, valganciclovir, Tenofovir, Adefovir,cidofovir, Efavirenz, Nevirapine, Delavirdine, and Etravirine.

In another embodiment, the additional therapeutic agent is aTLR-agonist. In a preferred embodiment, the TLR-agonist is selected fromthe group consisting of SM360320(9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848 (methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

In a further embodiment of the combination therapy, the additionaltherapeutic agent is an interferon, wherein the interferon is anyinterferon, which may be optionally pegylated. In yet a furtherembodiment, the therapeutic agent is an interferon selected from thegroup consisting of interferon alpha (IFN-α), interferon beta (IFN-β),interferon lambda (IFN-λ), and interferon gamma (IFN-γ). In a preferredembodiment, the interferon is interferon-alpha-2a, interferon-alpha-2b,interferon-alpha-n1, pegylated interferon-alpha-2a, or pegylatedinterferon-alpha-2b. In another preferred embodiment, the interferon isinterferon-alpha-2a, interferon-alpha-2b, or interferon-alpha-n1. In ayet another preferred embodiment, the interferon-alpha-2a orinterferon-alpha-2b is pegylated. In still another preferred embodiment,the interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS).

In another embodiment of the methods provided herein, administering thecompound of Formula I allows for administering of the at least oneadditional therapeutic agent at a lower dose or frequency as compared tothe administering of the at least one additional therapeutic agent alonethat is required to achieve similar results in prophylactically treatingan HBV infection in an individual in need thereof.

In yet another embodiment of the methods provided herein, administeringthe compound of Formula I reduces the viral load in the individual to agreater extent or at a faster rate compared to the administering of acompound selected from the group consisting of a HBV polymeraseinhibitor, interferon, viral entry inhibitor, viral maturationinhibitor, distinct capsid assembly modulator, antiviral compounds, andany combination thereof.

In still another embodiment of the methods provided herein,administering the compound of Formula I causes a lower incidence ofviral mutation or viral resistance than the administering of a compoundselected from the group consisting of a HBV polymerase inhibitor, aninterferon, a viral entry inhibitor, a viral maturation inhibitor, acapsid assembly modulator, an antiviral compound, and combinationsthereof.

In any of the methods provided herein, the method may further compriseadministering to the individual at least one HBV vaccine, a nucleosideHBV inhibitor, an interferon or any combination thereof. In anembodiment, the HBV vaccine is selected from the group consisting ofRECOMBIVAX HB, ENGERIX-B, ELOVAC B, GENEVAC-B, and SHANVAC B.

In another aspect, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising reducing the HBVviral load by administering to the individual a therapeuticallyeffective amount of a compound of Formula I alone or in combination witha reverse transcriptase inhibitor; and further administering to theindividual a therapeutically effective amount of HBV vaccine. In anembodiment, the reverse transcriptase inhibitor is at least one ofZidovudine, Didanosine, Zalcitabine, 2′,3′-dideoxyadenosine, Stavudine,Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine,Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, valganciclovir, Tenofovir, Adefovir, cidofovir, Efavirenz,Nevirapine, Delavirdine, and Etravirine.

In another embodiment of the methods provided herein, the method furthercomprises monitoring the HBV viral load of the subject, and wherein themethod is carried out for a period of time such that the HBV virus isundetectable.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that are useful in the treatment andprevention of HBV infection in man. In a non-limiting aspect, thesecompounds may modulate or disrupt HBV assembly and other HBV coreprotein functions necessary for the generation of infectious particlesby interacting with HBV capsid to afford defective viral particles withgreatly reduced virulence. The compounds of the invention have potentantiviral activity, exhibit favorable metabolic, tissue distribution,safety and pharmaceutical profiles, and are suitable for use in man.

The HBV capsid protein plays essential functions during the viral lifecycle. HBV capsid/core proteins form metastable viral particles orprotein shells that protect the viral genome during intercellularpassage, and also play a central role in viral replication processes,including genome encapsidation, genome replication, and virionmorphogenesis and egress. Capsid structures also respond toenvironmental cues to allow un-coating after viral entry. Consistently,proper capsid assembly and function of core protein have been found tobe critical for viral infectivity.

The crucial function of HBV capsid proteins imposes stringentevolutionary constraints on the viral capsid protein sequence, leadingto the observed low sequence variability and high conservation.Consistently, mutations in HBV capsid that disrupt its assembly arelethal, and mutations that perturb capsid stability severely attenuateviral replication. The more conserved a drug target is, the fewerreplication-competent resistance mutations are acquired by patients.Indeed, natural mutations in HBV capsid for chronically infectedpatients accumulate in only four out of 183 residues in the full lengthprotein. Thus, HBV capsid assembly and function inhibitors may elicitlower drug resistance emergence rates relative to existing HBVantivirals. Further, drug therapies that target HBV capsid could be lessprone to drug-resistant mutations when compared to drugs that targettraditional neuraminidase enzyme active sites. Reports describingcompounds that bind viral capsids and inhibit replication of HIV,rhinovirus and HBV provide strong pharmacological proof of concept forviral capsid proteins as antiviral drug targets.

In one aspect, the compounds of the invention are useful in HBVtreatment by disrupting, accelerating, reducing, delaying and/orinhibiting normal viral capsid assembly and/or disassembly of immatureor mature particles, thereby inducing aberrant capsid morphology andleading to antiviral effects such as disruption of virion assemblyand/or disassembly, virion maturation, and/or virus egress. In oneembodiment, a disruptor of capsid assembly interacts with mature orimmature viral capsid to perturb the stability of the capsid, thusaffecting assembly and/or disassembly. In another embodiment, adisruptor of capsid assembly perturbs protein folding and/or saltbridges required for stability, function and/or normal morphology of theviral capsid, thereby disrupting and/or accelerating capsid assemblyand/or disassembly. In yet another embodiment, the compounds of theinvention bind capsid and alter metabolism of cellular polyproteins andprecursors, leading to abnormal accumulation of protein monomers and/oroligomers and/or abnormal particles, which causes cellular toxicity anddeath of infected cells. In another embodiment, the compounds of theinvention cause failure of the formation of capsid of optimal stability,affecting efficient uncoating and/or disassembly of viruses (e.g.,during infectivity).

In one embodiment, the compounds of the invention disrupt and/oraccelerate capsid assembly and/or disassembly when the capsid protein isimmature. In another embodiment, the compounds of the invention disruptand/or accelerate capsid assembly and/or disassembly when the capsidprotein is mature. In yet another embodiment, the compounds of theinvention disrupt and/or accelerate capsid assembly and/or disassemblyduring viral infectivity. In yet another embodiment, the disruptionand/or acceleration of capsid assembly and/or disassembly attenuates HBVviral infectivity and/or reduces viral load. In yet another embodiment,disruption, acceleration, inhibition, delay and/or reduction of capsidassembly and/or disassembly eradicates the virus from the host organism.In yet another embodiment, eradication of the HBV from a hostadvantageously obviates the need for chronic long-term therapy and/orreduces the duration of long-term therapy.

In one embodiment, the compounds described herein are suitable formonotherapy and are effective against natural or native HBV strains andagainst HBV strains resistant to currently known drugs. In anotherembodiment, the compounds described herein are suitable for use incombination therapy.

In another embodiment, the compounds of the invention can be used inmethods of modulating (e.g., inhibit or disrupt) the activity,stability, function, and viral replication properties of HBV cccDNA. Inyet another embodiment, the compounds of the invention can be used inmethods of diminishing or preventing the formation of HBV cccDNA.

In another embodiment, the compounds of the invention can be used inmethods of modulating (e.g., inhibit or disrupt) the activity of HBVcccDNA. In yet another embodiment, the compounds of the invention can beused in methods of diminishing or preventing the formation of HBVcccDNA.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and peptide chemistryare those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e. to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, more preferably ±5%,even more preferably ±1%, and still more preferably ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, the term “capsid assembly modulator” refers to acompound that disrupts or accelerates or inhibits or hinders or delaysor reduces or modifies normal capsid assembly (e.g., during maturation)or normal capsid disassembly (e.g., during infectivity) or perturbscapsid stability, thereby inducing aberrant capsid morphology andfunction. In one embodiment, a capsid assembly modulator acceleratescapsid assembly or disassembly, thereby inducing aberrant capsidmorphology. In another embodiment, a capsid assembly modulator interacts(e.g. binds at an active site, binds at an allosteric site, modifiesand/or hinders folding and the like) with the major capsid assemblyprotein (CA), thereby disrupting capsid assembly or disassembly. In yetanother embodiment, a capsid assembly modulator causes a perturbation instructure or function of CA (e.g., ability of CA to assemble,disassemble, bind to a substrate, fold into a suitable conformation, orthe like), which attenuates viral infectivity and/or is lethal to thevirus.

As used herein, the term “treatment” or “treating,” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has HBV infection, a symptom ofHBV infection or the potential to develop HBV infection, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect HBV infection, the symptoms of HBV infection or thepotential to develop HBV infection. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the patient, subject or individual is human.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts of thepresent invention include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁₋₆means one to six carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁₋₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e., skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having 3 to 10 ring atoms (C₃₋₁₀cycloalkyl), or groups having 3 to 7 ring atoms (C₃₋₇ cycloalkyl).

The term cycloalkyl includes “unsaturated nonaromatic carbocyclyl” or“nonaromatic unsaturated carbocyclyl” groups, both of which refer to anonaromatic carbocycle as defined herein, which contains at least onecarbon carbon double bond or one carbon carbon triple bond.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S. The heteroatom(s) may be placed at any position of theheteroalkyl group, including between the rest of the heteroalkyl groupand the fragment to which it is attached, as well as attached to themost distal carbon atom in the heteroalkyl group. Examples include:—O—CH₂—CH₂—CH₃, —CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, and —CH₂—S—CH₂—CH₃. Upto two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃. Preferred heteroalkyl groups have1-10 carbons.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e., having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl. In some embodiments, arylgroups have six carbon atoms. In some embodiments, aryl groups have fromsix to ten carbon atoms. In some embodiments, aryl groups have from sixto sixteen carbon atoms.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. In some embodiments, heteroarylor heteroaromatic groups have two to five carbon atoms. In someembodiments, heteroaryl or heteroaromatic groups have from two to tencarbon atoms. In some embodiments, heteroaryl or heteroaromatic groupshave from two to sixteen carbon atoms. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. In someembodiments, polycyclic heteroaryl groups have two to five carbon atoms.In some embodiments, polycyclic heteroaryl groups have from two to tencarbon atoms. In some embodiments, polycyclic heteroaryl groups havefrom two to sixteen carbon atoms.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

Compounds of the Invention

The present invention relates to the discovery of compounds that areuseful in the treatment and prevention of HBV infection in man. In oneaspect, the compounds of the invention are useful in HBV treatment bydisrupting, accelerating, reducing delaying or inhibiting normal viralcapsid assembly or disassembly of immature or mature particles, therebyinducing aberrant capsid morphology and leading to antiviral effectssuch as disruption of virion assembly or disassembly or virionmaturation, or virus egress.

In another aspect, compounds of the invention bind to core proteinthereby inducing aberrant virion and leading to antiviral effects suchas disruption of virion assembly, disassembly, maturation, or virusegress.

The capsid assembly disruptors disclosed herein may be used asmonotherapy or in cross-class combination regimens for treating HBVinfection in man. Combination therapy with drugs exhibiting differentmechanism of action (MOA) that act at different steps in the virus lifecycle may deliver greater efficacy due to additive or synergisticantiviral effects. Clinically evaluated HIV treatment regimens haveshown that combination therapy improves the efficacy of viral loadreduction, and dramatically reduces emergence of antiviral resistance.Combination therapy for the treatment of Hepatitis C (HCV) virusinfection has also resulted in significant improvement in sustainedantiviral response and eradication rates. Thus, use of the HBV capsidassembly inhibitors of the present invention in combination with, forexample, neuraminidase drugs, is likely to deliver a more profoundantiviral effect and greater disease eradication rates than currentstandards of care.

Capsid assembly plays a central role in HBV genome replication. HBVpolymerase binds pre-genomic HBV RNA (pgRNA), and pgRNA encapsidationmust occur prior to HBV DNA synthesis. Moreover, it is well establishedthat nuclear accumulation of the cccDNA replication intermediate, whichis responsible for maintenance of chronic HBV replication in thepresence of nucleoside suppressive therapy, requires the capsid forshuttling HBV DNA to the nuclei. Therefore, the HBV capsid assemblydisruptors of the invention have the potential to increase HBVeradication rates through synergistic or additive suppression of viralgenome replication and to further reduce accumulation of cccDNA whenused alone or in combination with existing nucleoside drugs. The capsidassembly disruptors of the present invention may also alter normal coreprotein function or degradation, potentially leading to altered MHC-1antigen presentation, which may in turn increaseseroconversion/eradication rates through immuno-stimulatory activity,more effectively clearing infected cells.

In one aspect, drug resistance poses a major threat to current therapiesfor chronic HBV infection, and cross-class combination therapy is aproven strategy for delaying emergence of drug resistance strains. Thecapsid assembly disruptors of the present invention can, whenadministered alone or in combination with other HBV therapy, offerenhanced drug resistant profiles and improved management of chronic HBV.

The compounds useful within the invention can be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

In one aspect, the compound of the invention is a compound of Formula I:

or a pharmaceutically acceptable salt thereof;

wherein

R⁴ is H or C₁-C₃ alkyl;

R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, —H₂PO₄,—C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl,—C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, C₆-C₁₀ aryl, C₅-C₉heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄ alkyl-(C₅-C₉heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂;

R² is, independently at each occurrence, —OH, halo, —CN, —NO₂, R⁶, orOR⁶, wherein R⁶ is, independently at each occurrence, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, —C₃—C₁₀ heterocycloalkyl, C₆-C₁₀aryl, C₅-C₁₀ heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄alkyl-(C₃-C₁₀ heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄alkyl-(C₅-C₁₀ heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, —C₁-C₆alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl, —C₃-C₁₀cycloalkyl, —C₃-C₁₀ heterocycloalkyl, C₆-C₁₀ aryl, C₅-C₉ heteroaryl,—C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄ alkyl-(C₅-C₉heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂, or two R¹¹ groups, together withthe carbons to which they are attached, join to form a cyclic phosphatering;

m is 0, 1, 2, 3, or 4;

x is 0, 1, 2, 3, 4, or 5; and

y is 0, 1, 2, 3, or 4. In one embodiment, y is 0, 1, or 2.

In an embodiment of Formula I provided herein, or a pharmaceuticallyacceptable salt thereof,

R⁴ is H;

m is 0, 1, 2, or 3;

x is 0, 1, 2, or 3; and

y is 0, 1, 2, or 3. In a further embodiment, y is 0, 1, or 2. In yetanother embodiment, y is 1.

In another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, —C₁-C₆alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl, —C₃-C₁₀cycloalkyl, —C₃-C₁₀ heterocycloalkyl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl),or —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl), wherein the alkyl group isoptionally substituted 1-5 times with halo or —OH.

In yet another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R² is, independently at each occurrence, —OH, halo, —CN, —NO₂, R⁶, orOR⁶, wherein R⁶ is, independently at each occurrence, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, —C₁-C₄alkyl-(C₃-C₁₀ cycloalkyl), or —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl),wherein the alkyl group is optionally substituted 1-5 times with halo or—OH.

In still another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R¹¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, —C₁-C₆alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl, —C₃-C₁₀cycloalkyl, —C₃-C₁₀ heterocycloalkyl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl),or —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl), wherein the alkyl group isoptionally substituted 1-5 times with halo or —OH.

In another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, or —C₃-C₁₀ heterocycloalkyl.

In yet another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R⁴ is H;

each R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, or—C₁-C₆ alkyl;

R² is selected from —OH, halo, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₁₀cycloalkyl, and —C₃-C₁₀ heterocycloalkyl, wherein the alkyl andcycloalkyl groups are optionally substituted 1-5 times with halo;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH or halo;

m is 0, 1 or 2; and

x is 0, 1, 2, or 3.

In still another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R⁴ is H;

each R¹ is, independently at each occurrence, —OH or halo;

R² is selected from —OH, halo, and —C₁-C₆ alkyl, wherein the alkyl groupis optionally substituted 1-5 times with halo;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, or —C₃-C₁₀ heterocycloalkyl;

m is 0, 1 or 2; and

x is 0, 1, 2, or 3.

In another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R⁴ is H;

each R¹ is, independently at each occurrence, —OH or halo;

R² is selected from halo and —C₁-C₃ alkyl, wherein the alkyl group isoptionally substituted 1-3 times with halo;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃ alkyl,—C₁-C₄ heteroalkyl, —C₃-C₇ cycloalkyl, or —C₃-C₇ heterocycloalkyl;

m is 0, 1 or 2; and

x is 0, 1, 2, or 3.

In yet another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R⁴ is H;

each R¹ is, independently at each occurrence, halo;

R² is selected from halo and —C₁ alkyl, wherein the alkyl group isoptionally substituted 1-3 times with halo;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃ alkyl, or—C₃-C₇ cycloalkyl;

m is 0, 1 or 2; and

x is 2 or 3.

In still another embodiment of Formula I provided herein, or apharmaceutically acceptable salt thereof,

R⁴ is H;

each R¹ is, independently at each occurrence, halo;

R² is selected from halo and —C₁ alkyl, wherein the alkyl group isoptionally substituted 1-3 times with halo;

Cy is

wherein

R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃ alkyl, or—C₃-C₇ cycloalkyl;

m is 0, 1 or 2; and

x is 2 or 3.

In another embodiment of Formula I provided herein, the compound isselected from:

or a pharmaceutically acceptable salt thereof.

Certain preferred embodiments of Formula I, including pharmaceuticallyacceptable salts thereof, are shown below in Table 1. All compounds ofFormula I, as well as pharmaceutically acceptable salts thereof, and thecompounds of Table 1, as well as pharmaceutically acceptable saltsthereof, are considered to be “compounds of the invention.”

Synthetic method codes refer to the synthesis methodologies provided inthe experimental section. For example, “A01B01C01” refers the use ofintermediate A01 for region A, intermediate B01 for region B, andintermediate C01 for region C.

TABLE 1 Synthetic Structure Compound ID ¹H NMR MS (M + H)⁺ method

2037 443/445 A01B01C01

2038 445/447 A16B01C01

2039 ¹H NMR (400 MHz, MeOD) δ 8.46 (dd, J = 2.3, 6.5 Hz, 1H), 8.24 (m,1H), 7.95 (dd, J = 2.5, 6.5 Hz, 1H), 7.66-7.57 (m, 1H), 7.50 (t, J = 9.3Hz, 1H), 7.24 (t, J = 9.0 Hz, 1H), 4.01-4.17 (m, 2H), 3.79- 3.87 (m,1H), 3.56-3.77 (m, 4H), 3.10-3.23 (m, 2H), 2.18-2.29 (m, 1H), 1.68-1.79(m, 1H). 461/463 A17B01C01

2039_E1 ¹H NMR (400 MHz, MeOD) δ 8.46 (d, J = 5.3 Hz, 1H), 8.24 (brs,1H), 7.96 (d, J = 5.3 Hz, 1H), 7.61 (brs, 1H), 7.51 (t, J = 8.8 Hz, 1H),7.25 (t, J = 8.3 Hz, 1H), 3.97-4.22 (m, 2H), 3.51-3.92 (m, 5H), 3.06-3.25 (m, 2H), 2.23 (brs, 1H), 1.75 (brs, 1H). 461/463 A17B01C01Enantiomers were separated by SUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD-3S_2_5_40_3 ML_T35.M

2039_E2 ¹H NMR (400 MHz, MeOD) δ 8.46 (dd, J = 2.3, 6.5 Hz, 1H), 8.20-8.30 (m, 1H), 7.96 (dd, J = 2.4, 6.7 Hz, 1H), 7.59-7.65 (m, 1H), 7.51(t, J = 9.4 Hz, 1H), 7.25 (t, J = 8.9 Hz, 1H), 4.02-4.19 (m, 2H), 3.80-3.87 (m, 1H), 3.55-3.78 (m, 4H), 3.09-3.22 (m, 2H), 2.17- 2.30 (m, 1H),1.67-1.79 (m, 1H). 461/463 A17B01C01 Enantiomers were separated bySUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD- 3S_2_5_40_3 ML_T35.M

2040 ¹H NMR (400 MHz, MeOD) δ 8.44 (dd, J = 2.0, 6.5 Hz, 1H), 8.24 (m,1H), 7.96 (dd, J = 2.5, 6.5 Hz, 1H), 7.56-7.66 (m, 1H), 7.49 (t, J = 9.3Hz, 1H), 7.25 (t, J = 8.9 Hz, 1H), 3.71-3.98 (m, 5H), 3.39- 3.54 (m,3H), 3.32-3.37 (m, 1H), 1.95-2.15 (m, 2H). 461/463 A18B01C01

2040_E1 ¹H NMR (400 MHz, MeOD) δ 8.44 (dd, J = 2.0, 6.5 Hz, 1H), 8.18-8.28 (m, 1H), 7.96 (dd, J = 2.5, 6.5 Hz, 1H), 7.57-7.67 (m, 1H), 7.50(t, J = 9.3 Hz, 1H), 7.25 (t, J = 8.9 Hz, 1H), 3.71-3.99 (m, 5H), 3.39-3.54 (m, 3H), 3.35 (brs, 1H), 1.90-2.17 (m, 2H). 461/463 A18B01C01Enantiomers were separated by SUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD-3S_2_5_40_3 ML_T35.M

2040_E2 ¹H NMR (400 MHz, MeOD) δ 8.44 (dd, J = 2.0, 6.5 Hz, 1H), 8.19-8.29 (m, 1H), 7.96 (dd, J = 2.5, 6.8 Hz, 1H), 7.57-7.66 (m, 1H), 7.50(t, J = 9.3 Hz, 1H), 7.25 (t, J = 9.0 Hz, 1H), 3.71-3.98 (m, 5H), 3.42-3.54 (m, 3H), 3.35 (brs, 1H), 1.94-2.15 (m, 2H). 461/463 A18B01C01Enantiomers were separated by SUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD-3S_2_5_40_3 ML_T35.M

2069 473/475 A03B01C01

2070 ¹H NMR (400 MHz, MeOD) δ 8.46 (d, J = 6.5 Hz, 1H), 8.22-8.30 (m,1H), 7.47-7.67 (m, 3H), 3.35-3.43 (m, 3H), 3.06-3.19 (m, 1H), 1.50- 1.93(m, 8H), 1.26 (s, 3H). 475 A03B01C02

2227 455/457 (m − 18)⁺ A15B02C01

2228 ¹H NMR (400 MHz, MeOD) δ 8.19- 8.30 (m, 2H), 7.91 (d, J = 8.0 Hz,1H), 7.57-7.68 (m, 2H), 5.78-5.98 (m, 2H), 3.98-4.11 (m, 1H), 3.39-3.49(m, 2H), 3.21-3.30 (m, 2H), 1.66- 2.03 (m, 8H). 475 A15B02C02

2229 ¹H NMR (400 MHz, MeOD) δ 8.22- 8.30 (m, 2H), 7.90-8.01 (m, 2H),7.63-7.65 (m, 1H), 7.24- 7.31 (m, 1H), 5.97 (s, 1H), 5.81 (s, 1H),3.85-3.95 (m, 1H), 3.40- 3.50 (m, 4H), 1.78-2.00 (m, 8H). 473/475A04B02C01

2230 475 A15B02C02

2231 473/475 A02B02C01

2232 475 A02B02C02

2233 457/459 A01B02C01

2234 459 A02B02C02

2237 475/477 A17B02C01

2238 477 A17B02C02

2239 475/477 A18B02C01

2240 477 A18B02C02

2241 471/473 (M − 18)⁺ A19B02C01

2242 ¹H NMR (400 MHz, MeOD) δ 8.32- 8.37 (m, 1 H), 8.20-8.27 (m, 1 H),7.89-7.94 (m, 1 H), 7.56- 7.66 (m, 2 H), 5.94-5.98 (m, 1 H), 5.82-5.86(m, 1 H), 3.64- 3.85 (m, 4 H), 3.54-3.61 (m, 1 H), 3.37-3.51 (m, 2 H),3.16- 3.25 (m, 1 H), 2.07-2.17 (m, 1 H), 1.67-1.77 (m, 1 H), 1.26 (s, 3H). 473/513 (M − 18)⁺/ (M + 23)⁺ A19B02C02

2249 469/471 (M − 18)⁺ A03B02C01

2250 471 (M − 18)⁺ A03B02C02

2253 469/471 (M − 18)⁺ A05B02C01

2254 471 (M − 18)⁺ A05B02C02

2255 495/497 (M − 18)⁺ A06B02C01

2256 497 (M − 18)⁺ A06B02C02

2257 469/471 (M − 18)⁺ A08B02C01

2258 471 (M − 18)⁺ A08B02C02

2259 495/497 (M − 18)⁺ A09B02C01

2260 ¹H NMR (400 MHz, MeOD) δ 8.29 (s, 1 H), 8.22 (dd, J = 8.1, 1.6 Hz,1 H), 7.91 (d, J = 8.0 Hz, 1 H), 7.56-7.66 (m, 2 H), 5.77-5.98 (m, 2 H),3.34-3.52 (m, 4 H), 1.64-2.06 (m, 8 H), 0.92-1.10 (m, 1 H), 0.26-0.46(m, 4 H). 497 (M − 18)⁺ A09B02C02

2261 457/459 (M − 18)⁺ A19B01C01

2262 459/499 (M − 18)⁺/ (M + 23)⁺ A19B01C02

2273 ¹H NMR (400 MHz, MeOD) δ 8.39- 8.48 (m, 1 H), 8.18-8.27 (m, 1 H),7.93-8.01 (m, 1 H), 7.57- 7.67 (m, 1 H), 7.45-7.52 (m, 1 H), 7.20-7.31(m, 1 H), 3.41- 3.63 (m, 2 H), 3.03-3.15 (m, 2 H), 1.98-2.13 (m, 1 H),1.87- 1.97 (m, 2 H), 1.48-1.86 (m, 5 H), 1.27 (s, 3 H). 455/457 (M −18)⁺ A05B01C01

2274 457 (M − 18)⁺ A05B01C02

2275 481/483 (M − 18)⁺ A06B01C01

2276 ¹H NMR (400 MHz, MeOD) δ 8.38- 8.50 (m, 1 H), 8.20-8.27 (m, 1 H),7.55-7.65 (m, 2 H), 7.45- 7.54 (m, 1 H), 3.50-3.73 (m, 2 H), 3.00-3.19(m, 2 H), 1.96- 2.13 (m, 2 H), 1.59-1.94 (m, 6 H), 1.04-1.19 (m, 1 H),0.41- 0.51 (m, 1H), 0.26-0.40 (m, 3 H). 483 (M − 18)⁺ A06B01C02

2277 ¹H NMR (400 MHz, MeOD) δ 10.68 (s, 1 H), 8.39-8.40 (m, 1 H),8.35-8.37 (m, 1 H), 8.03- 8.06 (m, 1 H), 7.68-7.71 (m, 2 H), 7.43-7.48(m, 1 H), 4.21 (s, 1 H), 3.18-3.34 (m, 4 H), 1.53- 1.79 (m, 8 H), 1.10(s, 3 H). 455/457 (M − 18)⁺ A08B01C01

2278 457 (M − 18)⁺ A08B01C02

2279 ¹H NMR (400 MHz, MeOD) δ 8.44- 8.47 (m, 1 H), 8.23-8.29 (m, 1 H),7.97-7.99 (m, 1 H), 7.55- 7.65 (m, 1 H), 7.48-7.53 (m, 1 H), 7.25-7.29(m, 1 H), 3.25- 3.50 (m, 4 H), 1.76-1.96 (m, 8 H), 0.98-1.02 (m, 1 H),0.33- 0.41 (m, 4 H). 481/483 (M − 18)⁺ A09B01C01

2280 483 (M − 18)⁺ A09B01C02

2285_D1 ¹H NMR (400 MHz, MeOD) δ 8.46- 8.52 (m, 1 H), 8.23-8.32 (m, 1H), 7.95-8.02 (m, 1 H), 7.61- 7.68 (m, 1 H), 7.49-7.59 (m, 1 H),7.21-7.32 (m, 1 H), 4.39- 4.65 (m, 1 H), 3.71-3.89 (m, 2 H), 3.49-3.61(m, 1 H), 3.34- 3.42 (m, 2 H), 3.10 (d, J = 13.6 Hz, 1 H), 1.69-2.06 (m,6 H). 477/479 A14B01C01

2285_D2 ¹H NMR (400 MHz, MeOD) δ 8.45-8.53 (m, 1 H), 8.23-8.32 (m, 1 H),7.94-8.01 (m, 1 H), 7.61-7.69 (m, 1 H), 7.48-7.59 (m, 1 H), 7.22-7.31(m, 1 H), 4.93-4.95 (m, 1 H), 4.75-4.86 (m, 1 H), 4.10-4.28 (m, 1 H),3.68-3.82 (m, 1 H), 3.39-3.62 (m, 2 H), 2.98-3.13 (m, 1H), 2.06 (s, 6H). 477/479 A14B01C01

2286_D1 ¹H NMR (400 MHz, MeOD) δ 8.43-8.53 (m, 1 H), 8.23-8.34 (m, 1 H),7.45-7.68 (m, 3 H), 4.41-4.65 (m, 1 H), 3.67-3.88 (m, 2 H), 3.50-3.59(m, 1 H), 3.34-3.43 (m, 1 H), 3.04-3.17 (m, 1 H), 1.67-2.06 (m, 6 H).479 A14B01C02

2286_D2 ¹H NMR (400 MHz, MeOD) δ 8.45-8.50 (m, 1 H), 8.24-8.31 (m, 1 H),7.49-7.66 (m, 3 H), 4.78-4.83 (m, 1 H), 4.11-4.26 (m, 1H), 3.67-3.78 (m,1 H), 3.42-3.62 (m, 2H), 3.03-3.13 (m, 1H), 1.63-2.07 (m, 6 H). 479A14B01C02

2287_D1 491/493 A14B02C01

2287_D2 491/493 A14B02C01

2288_D1 493 A14B02C02

2288_D2 493 A14B02C02

2293_D1 477/479 A12B01C01

2293_D2 477/479 A12B01C01

2294_D1 ¹H NMR (400 MHz, MeOD) δ 8.45-8.47 (m, 1 H), 8.24-8.26 (m, 1 H),7.49-7.63 (m, 3 H), 4.53-4.65 (m, 1 H), 4.19-4.24 (m, 1 H), 3.74-3.76(m, 1 H), 3.52-3.56 (m, 1 H), 3.03-3.09 (m, 2 H), 1.82-2.13 (m, 6 H).479 A12B01C02

2294_D2 ¹H NMR (400 MHz, MeOD) δ 8.44-8.46 (m, 1 H), 8.23-8.26 (m, 1 H),7.51-7.63 (m, 3 H), 4.79-4.92 (m, 1 H), 4.26-4.32 (m, 1 H), 3.57-3.66(m, 2 H), 3.10-3.20 (m, 1 H), 2.99-3.04 (m, 1 H), 1.75-2.30 (m, 6 H).479 A12B01C02

2297_Trans1 ¹H NMR (400 MHz, MeOD) δ 8.47 (dd, J = 2.0, 6.5 Hz, 1 H),8.22-8.32 (m, 1 H), 7.97 (dd, J = 2.5, 6.5 Hz, 1 H), 7.67-7.58 (m, 1 H),7.53 (t, J = 9.3 Hz, 1 H), 7.25 (t, J = 8.9 Hz, 1 H), 4.28-4.48 (m, 1H), 4.08-4.26 (m, 2 H), 4.01 (dt, J = 5.8, 12.3 Hz, 1 H), 3.70- 3.90 (m,2 H), 3.44-3.59 (m, 2 H), 3.33-3.37 (m, 1 H), 3.27 (brs, 1 H). 479/481A21B01C01 Regiomers were separated by SUPER- CRITICAL FLUID CHOMATO-GRAPHY: AD- 3S_5_5_40_3 ML_T35.M

2297_Trans2 ¹H NMR (400 MHz, MeOD) δ 8.47 (dd, J = 2.0, 6.5 Hz, 1 H),8.26 (dt, J = 2.3, 5.4 Hz, 1H), 7.96 (dd, J = 2.5, 6.5 Hz, 1 H), 7.62(td, J = 3.3, 8.9 Hz, 1 H), 7.52 (t, J = 9.4 , 1 H), 7.25 (t, J = 9.0Hz, 1 H), 4.28-4.48 (m, 1 H), 4.10-4.27 (m, 2H), 4.01 (dt, J = 5.6, 12.4Hz, 1 H), 3.71-3.89 (m, 2 H), 3.44-3.58 (m, 2 H), 3.33-3.37 (m, 1 H),3.26-3.30 (m, 1H). 479/481 A21B01C01 Regiomers were separated by SUPER-CRITICAL FLUID CHOMATO- GRAPHY: AD- 3S_5_5_40_3 ML_T35.M

2301_Trans1 ¹H NMR (400 MHz, MeOD) δ 8.48 (dd, J = 2.3, 6.8 Hz, 1 H),8.27 (ddd, J = 2.3, 4.7, 8.6 Hz, 1 H), 7.97 (dd, J = 2.5, 6.5 Hz, 1 H),7.59-7.65 (m, 1 H), 7.49-7.57 (m, 1 H), 7.25 (t, J = 8.9 Hz, 1 H), 4.69(d, J = 3.0 Hz, 1 H), 4.57 (d, J = 3.3 Hz, 1 H), 3.95-4.03 (m, 1 H),3.81-3.93 (m, 2H), 3.68- 3.80 (m, 2 H), 3.55-3.67 (m, 2 H), 3.49 (d, J =14.3 Hz, 1 H), 3.27 (brs, 1 H). 479/481 A22B01C01 Regiomers wereseparated by SUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD- 3S_5_5_40_3ML_T35.M

2301_Trans2 ¹H NMR (400 MHz, MeOD) δ 8.48 (dd, J = 2.3, 6.8 Hz, 1 H),8.27 (m, 1 H), 7.97 (dd, J = 2.5, 6.5 Hz, 1 H), 7.58-7.67 (m, 1 H), 7.53(dd, J = 8.9, 9.9 Hz, 1 H), 7.25 (t, J = 8.9 Hz, 1 H), 4.69 (dt, J =2.9, 8.3 Hz, 1 H), 4.57 (d, J = 3.0 Hz, 1 H), 3.99 (m, 1 H), 3.81- 3.93(m, 2 H), 3.68-3.80 (m, 2 H), 3.55-3.67 (m, 2 H), 3.49 (d, J = 14.3 Hz,1 H), 3.25-3.29 (m, 1 H). 479/481 A22B01C01 Regiomers were separated bySUPER- CRITICAL FLUID CHOMATO- GRAPHY: AD- 3S_5_5_40_3 ML_T35.M

2309_D1 473/475 A10B01C01

2309_D2 473/475 A10B01C01

2310_D1 ¹H NMR (400 MHz, MeOD) δ 8.44-8.47 (m, 1 H), 8.24-8.26 (m, 1 H),7.52-7.63 (m, 3 H), 4.01-4.03 (m, 1 H), 3.44-3.54 (m, 2 H), 3.09-3.15(m, 2 H), 2.10-2.13 (m, 1 H), 1.69-1.79 (m, 5 H), 1.47-1.63 (m, 1 H),1.04-1.06 (m, 3 H). 475 A10B01C02

2310_D2 ¹H NMR (400 MHz, MeOD) δ 8.45-8.47 (m, 1 H), 8.23-8.26 (m, 1 H),7.50-7.64 (m, 3 H), 3.48-3.63 (m, 3 H), 2.96-3.03 (m, 2 H), 1.56-2.06(m, 7 H), 1.09-1.11 (m, 3 H). 475 A10B01C02

2311_D1 487/489 A10B02C01

2311_D2 487/489 A10B02C01

2312_D1 ¹H NMR (400 MHz, MeOD) δ 8.32 (s, 1 H), 8.23-8.26 (m, 1 H),7.92-7.94 (m, 1 H), 7.61-7.65 (m, 2 H), 5.95 (s, 1 H), 5.83 (s, 1 H),3.96-4.00 (m, 1 H), 3.47- 3.54 (m, 2 H), 3.15-3.21 (m, 2 H), 2.06-2.12(m, 1 H), 1.69- 1.81 (m, 5 H), 1.45-1.47 (m, 1 H), 1.03-1.05 (m, 3 H).489 A10B02C02

2312_D2 ¹H NMR (400 MHz, MeOD) δ 8.32 (s, 1 H), 8.22-8.25 (m, 1 H),7.92-7.94 (m, 1 H), 7.61-7.65 (m, 2 H), 5.96 (s, 1 H), 5.84 (s, 1 H),3.48-3.60 (m, 3 H), 3.07- 3.12 (m, 2 H), 1.59-1.92 (m, 7 H), 1.08-1.10(m, 3 H). 489 A10B02C02

2313_D1 ¹H NMR (400 MHz, MeOD) δ 8.43-8.46 (m, 1 H), 8.20-8.25 (m, 1 H),7.80-7.97 (m, 1 H), 7.62- 7.65 (m, 1 H), 7.49-7.53 (m, 1 H), 7.25-7.29(m, 1 H), 3.25- 3.40 (m, 3 H), 3.10-3.13 (m, 2 H), 2.04-2.14 (m, 3 H),1.70- 1.81 (m, 4 H), 1.01-1.03 (m, 3 H). 473/475 A11B01C01

2313_D2 ¹H NMR (400 MHz, MeOD) δ 8.44-8.47 (m, 1 H), 8.20-8.25 (m, 1 H),7.80-7.97 (m, 1 H), 7.60- 7.65 (m, 1 H), 7.48-7.53 (m, 1 H), 7.24-7.29(m, 1 H), 3.94- 3.97 (m, 1 H), 3.26-3.40 (m, 3 H), 3.08-3.11 (m, 1 H),2.30- 2.35 (m, 1 H), 1.60-2.06 (m, 6 H), 0.96-0.98 (m, 3 H). 473/475A11B01C01

2314_D1 475 A11B01C02

2314_D2 475 A11B01C02

2315_D1 487/489 A11B02C01

2315_D2 487/489 A11B02C01

2316_D1 489 A11B02C02

2316_D2 ¹H NMR (400 MHz, MeOD) δ 8.29 (s, 1 H), 8.21-8.24 (m, 1 H),7.91-7.93 (m, 1 H), 7.61-7.65 (m, 2 H), 5.94 (s, 1 H), 5.83 (s, 1 H),3.95-3.97 (m, 1 H), 3.32- 3.42 (m, 3 H), 3.10-3.14 (m, 1 H), 2.30-2.35(m, 1 H), 1.60- 1.94 (m, 6 H), 0.96-0.98 (m, 3 H). 489 A11B02C02

2317_D1 473/475 A07B01C01

2317_D2 473/475 A07B01C01

2318_D1 475 A07B01C02

2318_D2 475 A07B01C02

2319_D1 487/489 A07B02C01

2319_D2 487/489 A07B02C01

2320_D1 489 A07B02C02

2320_D2 489 A07B02C02

2321_D1 ¹H NMR (400 MHz, MeOD) δ 8.44-8.46 (m, 1 H), 8.20-8.25 (m, 1 H),7.97-7.99 (m, 1 H), 7.60-7.65 (m, 1 H), 7.48-7.52 (m, 1 H), 7.25-7.29(m, 1 H), 3.72- 3.78 (m, 2 H), 3.50-3.55 (m, 1 H), 3.00-3.05 (m, 1 H),2.84- 2.88 (m, 1 H), 1.63-2.02 (m, 7 H), 1.18-1.20 (m, 3 H). 473/475A13B01C01

2321_D2 ¹H NMR (400 MHz, MeOD) δ 8.44-8.46 (m, 1 H), 8.20-8.25 (m, 1 H),7.97-7.99 (m, 1 H), 7.60-7.65 (m, 1 H), 7.48-7.52 (m, 1 H), 7.25-7.29(m, 1 H), 3.90- 3.92 (m, 1 H), 3.61-3.65 (m, 1 H), 3.48-3.50 (m, 1 H),3.05- 3.19 (m, 2 H), 1.52-2.08 (m, 7 H), 1.04-1.06 (m, 3 H). 473/475A13B01C01

2322_D1 475 A13B01C02

2322_D2 475 A13B01C02

2325 ¹H NMR (400 MHz, MeOD) δ 8.48 (dd, J = 2.3, 6.8 Hz, 1 H), 8.25 (m,1 H), 7.96 (dd, J = 2.6, 6.7 Hz, 1 H), 7.58-7.66 (m, 1 H), 7.52 (dd, J =9.0, 9.8 Hz, 1 H), 7.25 (t, J = 8.9 Hz, 1 H), 3.80- 3.93 (m, 2 H),3.67-3.76 (m, 1 H), 3.34-3.66 (m, 6 H), 1.87- 2.00 (m, 1 H), 0.97 (d, J= 6.8 Hz, 3H). 475/477 A20B01C01

2435 459 A27B01Co2

2436 457/459 A27B01C01

2448 ¹H NMR (400 MHz, MeOD) δ 8.45-8.43 (dd, 1 H), 8.22-8.24 (m, 1 H),7.95-7.97 (m, 1 H), 7.60-7.64 (m, 1 H), 7.47-7.52 (t, 1 H), 7.23-7.28(t, 1 H), 4.02 (m, 1 H), 3.33-3.37 (m, 2 H), 3.13- 3.17 (m, 2 H),1.83-1.97 (m, 8 H), 1.67-1.70 (m, 2 H). 473/475 A28B01C01

2483 ¹H NMR (400 MHz, MeOD) δ 8.47 (dd, J = 6.52, 2.01 Hz, 1 H),8.21-8.30 (m, 1 H), 7.47-7.67 (m, 3 H), 3.87-3.99 (m, 4 H), 3.76 (s, 4H), 3.43-3.54 (m, 4 H). 463 A26B01C02

2484 461/463 A26B01C01

2518 463/465 A17B01C02

2519 445/447 A17B01C03

2520 463/465 A18B01C02

2520_E1 463/465 A18B01C02 Enantiomers were separated by SUPER- CRITICALFLUID CHOMATO- GRAPHY

2520_E2 463/465 A18B01C02 Enantiomers were separated by SUPER- CRITICALFLUID CHOMATO- GRAPHY

2521 445/447 A18B01C03

2527 459 A17B02C03

2533 ¹H NMR (400 MHz, MeOD) δ 8.43-8.52 (m, 1 H), 8.22-8.32 (m, 1 H),7.76-7.91 (m, 1 H), 7.39-7.56 (m, 2 H), 7.22-7.32 (m, 1 H), 3.67-3.87(m, 4 H), 3.35-3.56 (m, 3 H), 3.12-3.24 (m, 1 H), 2.01-2.14 (m, 1 H),1.68-1.79 (m, 1 H), 1.26 (s, 3 H). 441 (M − 18)⁺ A19B01C03

2534 455/495 (M − 18)⁺/ (M + 23)⁺ A19B02C03

2584 ¹H NMR (400 MHz, MeOD) δ 8.46-8.48 (dd, 1 H), 8.22-8.26 (m, 1H),7.57-7.61 (dd, 2 H), 7.49-7.54 (dd., 1 H), 3.96-3.99 (m, 1 H), 3.87-3.91(dd, 1 H), 3.69-3.84 (m, 4 H), 3.58-3.62 (dd, 1 H), 3.07-3.15 (m, 2 H),2.05-2.14 (m, 1 H), 1.85-1.92 (m, 1 H). 463 A23B01C02

2580_Trans ¹H NMR (400 MHz, MeOD) δ 8.46-8.48 (dd, 1 H), 8.23-8.25 (m, 1H), 7.51-7.61 (m, 3 H), 4.03 (m, 1 H), 3.86-3.87 (m, 2 H), 3.68-3.73 (m,2 H), 3.54-3.57 (m, 3 H), 3.26-3.22 (m, 2 H). 479 A24B01C02

2580_Cis ¹H NMR (400 MHz, MeOD) δ 8.45-8.47 (dd, 1 H), 8.23-8.27 (m, 1H), 7.49-7.61 (m, 3 H), 4.10- 4.18 (m, 2H), 3.92-4.00 (m, 2 H),3.59-3.71 (m, 4 H), 3.26- 3.29 (m, 1 H), 3.06-3.12 (m, 1 H). 479A25B01C02

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the R or Sconfiguration. In one embodiment, compounds described herein are presentin optically active or racemic forms. It is to be understood that thecompounds described herein encompass racemic, optically-active,regioisomeric and stereoisomeric forms, or combinations thereof thatpossess the therapeutically useful properties described herein.

Preparation of optically active forms is achieved in any suitablemanner, including by way of non-limiting example, by resolution of theracemic form with recrystallization techniques, synthesis fromoptically-active starting materials, chiral synthesis, orchromatographic separation using a chiral stationary phase. In oneembodiment, a mixture of one or more isomer is utilized as thetherapeutic compound described herein. In another embodiment, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

In one embodiment, the compounds of the invention may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In another embodiment, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet anotherembodiment, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In one embodiment, the compounds described herein are labeled by othermeans, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey and Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green and Wuts,Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all ofwhich are incorporated herein in their entirety by reference for suchdisclosure). General methods for the preparation of compound asdescribed herein are modified by the use of appropriate reagents andconditions, for the introduction of the various moieties found in theformula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

In one embodiment, reactive functional groups, such as hydroxyl, amino,imino, thio or carboxy groups, are protected in order to avoid theirunwanted participation in reactions. Protecting groups are used to blocksome or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In another embodiment, each protective group is removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval.

In one embodiment, protective groups are removed by acid, base, reducingconditions (such as, for example, hydrogenolysis), and/or oxidativeconditions. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and are used to protect carboxy andhydroxy reactive moieties in the presence of amino groups protected withCbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties areblocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl, in the presence of amines that are blocked with acidlabile groups, such as t-butyl carbamate, or with carbamates that areboth acid and base stable but hydrolytically removable.

Methods of the Invention

The invention provides a method of treating an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention also provides a method of reducing the viral loadassociated with an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

The invention also provides a method of reducing reoccurrence of an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound of theinvention.

The invention further provides a method of reducing an adversephysiological impact of an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound of the invention.

The invention further provides a method of inducing remission of hepaticinjury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

The invention also provides a method of reducing the physiologicalimpact of long-term antiviral therapy for HBV infection in an individualin need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention also provides a method of prophylactically treating an HBVinfection in an individual in need thereof, wherein the individual isafflicted with a latent HBV infection, comprising administering to theindividual a therapeutically effective amount of a compound of theinvention.

In one embodiment, the methods described herein further compriseadministering to the individual at least one additional therapeuticagent selected from the group consisting of a HBV polymerase inhibitor,immunomodulatory agents, pegylated interferon, viral entry inhibitor,viral maturation inhibitor, capsid assembly modulator, reversetranscriptase inhibitor, a cyclophilin/TNF inhibitor, a TLR-agonist, andan HBV vaccine, and a combination thereof. In another embodiment, thecompound of the invention and the at least one additional therapeuticagent are co-formulated. In yet another embodiment, the compound of theinvention and the at least one additional therapeutic agent areco-administered.

In one embodiment, the administering a compound of the invention allowsfor administering of the at least one additional therapeutic agent at alower dose or frequency as compared to the administering of the at leastone additional therapeutic agent alone that is required to achievesimilar results in prophylactically treating an HBV infection in anindividual in need thereof.

In one embodiment, the administering of a compound of the inventionreduces the viral load in the individual to a greater extent or at afaster rate compared to the administering of a compound selected fromthe group consisting of a HBV polymerase inhibitor, interferon, viralentry inhibitor, viral maturation inhibitor, distinct capsid assemblymodulator, antiviral compounds, and any combination thereof.

In one embodiment, the administering of a compound of the inventioncauses a lower incidence of viral mutation or viral resistance than theadministering of a compound selected from the group consisting of a HBVpolymerase inhibitor, an interferon, a viral entry inhibitor, a viralmaturation inhibitor, a capsid assembly modulator, an antiviralcompound, and combinations thereof.

The invention also provide a method of treating an HBV infection in anindividual in need thereof, comprising reducing the HBV viral load byadministering to the individual a therapeutically effective amount of acompound of the invention alone or in combination with a reversetranscriptase inhibitor; and further administering to the individual atherapeutically effective amount of HBV vaccine.

In one embodiment, the method of the invention further comprisesmonitoring the HBV viral load of the subject, and wherein the method iscarried out for a period of time such that the HBV virus isundetectable.

Accordingly, in one embodiment, provided herein is a method of treatingan HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of Formula I, or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2039, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2039_E1,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2039_E2,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2040, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2040_E1,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2040_E2,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2285_D1,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2285_D2,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2435, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2436, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2520, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2520_E1,or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2520_E2,or a pharmaceutically acceptable salt thereof.

Combination Therapies

The compounds of the present invention are intended to be useful incombination with one or more additional compounds useful for treatingHBV infection. These additional compounds may comprise compounds of thepresent invention or compounds known to treat, prevent, or reduce thesymptoms or effects of HBV infection. Such compounds include but are notlimited to HBV polymerase inhibitors, interferons, viral entryinhibitors, viral maturation inhibitors, literature-described capsidassembly modulators, reverse transcriptase inhibitor, a TLR-agonist, andother agents with distinct or unknown mechanisms that affect the HBVlife cycle and/or affect the consequences of HBV infection.

In non-limiting examples, the compounds of the invention may be used incombination with one or more drugs (or a salt thereof) selected from thegroup consisting of:

HBV reverse transcriptase inhibitors, and DNA and RNA polymeraseinhibitors, including but not limited to: lamivudine (3TC, Zeffix,Heptovir, Epivir, and Epivir-HBV), entecavir (Baraclude, Entavir),adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA), tenofovirdisoproxil fumarate (Viread, TDF or PMPA);

interferons, including but not limited to interferon alpha (IFN-α),interferon lambda (IFN-λ), and interferon gamma (IFN-γ);

viral entry inhibitors;

viral maturation inhibitors;

literature-described capsid assembly modulators, such as, but notlimited to BAY 41-4109;

reverse transcriptase inhibitor;

a TLR-agonist; and

agents of distinct or unknown mechanism, such as but not limited toAT-61((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide),AT-130((E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide),and similar analogs.

In one embodiment, the additional therapeutic agent is an interferon.The term “interferon” or “IFN” refers to any member the family of highlyhomologous species-specific proteins that inhibit viral replication andcellular proliferation, and modulate immune response. Human interferonsare grouped into three classes; Type I, which include interferon-alpha(IFN-α), interferon-beta (IFN-β), and interferon-omega (IFN-ω), Type II,which includes interferon-gamma (IFN-γ), and Type III, which includesinterferon-lambda (IFN-λ). Recombinant forms of interferons that havebeen developed and are commercially available are encompassed by theterm “interferon” as used herein. Subtypes of interferons, such aschemically modified or mutated interferons, are also encompassed by theterm “interferon” as used herein. Chemically modified interferonsinclude pegylated interferons and glycosylated interferons. Examples ofinterferons include, but are not limited to, interferon-alpha-2a,interferon-alpha-2b, interferon-alpha-n1, interferon-beta-1a,interferon-beta-1b, interferon-lamda-1, interferon-lamda-2, andinterferon-lamda-3. Examples of pegylated interferons include pegylatedinterferon-alpha-2a and pegylated interferon alpha-2b.

Accordingly, in one embodiment, the compounds of Formula I can beadministered in combination with an interferon selected from the groupconsisting of interferon alpha (IFN-α), interferon beta (IFN-β),interferon lambda (IFN-λ), and interferon gamma (IFN-γ). In one specificembodiment, the interferon is interferon-alpha-2a, interferon-alpha-2b,or interferon-alpha-n1. In another specific embodiment, theinterferon-alpha-2a or interferon-alpha-2b is pegylated. In a preferredembodiment, the interferon-alpha-2a is pegylated interferon-alpha-2a(PEGASYS).

In another embodiment, the additional therapeutic agent is a reversetranscriptase inhibitor, and is at least one of Zidovudine, Didanosine,Zalcitabine, 2′,3′-dideoxyadenosine, Stavudine, Lamivudine, Abacavir,Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin,acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir,Tenofovir, Adefovir, cidofovir, Efavirenz, Nevirapine, Delavirdine, andEtravirine.

In one embodiment, the additional therapeutic agent is a TLR modulatoror a TLR agonist, such as a TLR-7 agonist or TLR-9 agonist. In a furtherembodiment of the combination therapy, the TLR agonist is selected fromthe group consisting of SM360320(9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848 (methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

In any of the methods provided herein, the method may further compriseadministering to the individual at least one HBV vaccine, a nucleosideHBV inhibitor, an interferon or any combination thereof. In anembodiment, the HBV vaccine is selected from the group consisting ofRECOMBIVAX HB, ENGERIX-B, ELOVAC B, GENEVAC-B, and SHANVAC B.

In another aspect, provided herein is method of treating an HBVinfection in an individual in need thereof, comprising reducing the HBVviral load by administering to the individual a therapeuticallyeffective amount of a compound of the invention alone or in combinationwith a reverse transcriptase inhibitor; and further administering to theindividual a therapeutically effective amount of HBV vaccine. Thereverse transcriptase inhibitor may be one of Zidovudine, Didanosine,Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir, Emtricitabine,Entecavir, Apricitabine, Atevirapine, ribavirin, acyclovir, famciclovir,valacyclovir, ganciclovir, valganciclovir, Tenofovir, Adefovir,cidofovir, Efavirenz, Nevirapine, Delavirdine, or Etravirine.

There can be three types of interactions between medications: additive,synergistic, and antagonistic. Additive interaction means the effect oftwo agents is equal to the sum of the effect of the two agents takenseparately at the same doses. Synergistic interaction means that theeffect of two agents taken together is greater than the sum of theirseparate effect at the same doses. Antagonistic interaction means thatthe effect of two agents is less than the sum of the effect of the twoagents taken independently of each other at the same doses.

For any combination therapy described herein, synergistic effect may becalculated, for example, using suitable methods such as theSigmoid-E_(max) equation (Holford & Scheiner, 19981, Clin.Pharmacokinet. 6: 429-453), the equation of Loewe additivity (Loewe &Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and themedian-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimentaldata to generate a corresponding graph to aid in assessing the effectsof the drug combination. The corresponding graphs associated with theequations referred to above are the concentration-effect curve,isobologram curve and combination index curve, respectively.

Administration/Dosage/Formulations

In another aspect, provided herein is pharmaceutical compositioncomprising a compound of the invention, or a pharmaceutically acceptablesalt thereof, together with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of HBV infection in a patient.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg to about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., another drug forHBV treatment) as described herein is less than about 1,000 mg, or lessthan about 800 mg, or less than about 600 mg, or less than about 500 mg,or less than about 400 mg, or less than about 300 mg, or less than about200 mg, or less than about 100 mg, or less than about 50 mg, or lessthan about 40 mg, or less than about 30 mg, or less than about 25 mg, orless than about 20 mg, or less than about 15 mg, or less than about 10mg, or less than about 5 mg, or less than about 2 mg, or less than about1 mg, or less than about 0.5 mg, and any and all whole or partialincrements thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof HBV infection in a patient.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

For oral application, particularly suitable are tablets, dragées,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For parenteral administration, the compounds of the invention may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials:

Unless otherwise noted, all starting materials and resins were obtainedfrom commercial suppliers and used without purification.

Region A:

Region B:

Region C:

Part I Intermediate Synthesis (Region A) 1.1 Preparation of Compound A02

1.1.1 Preparation of Compound 2A/2B

To a solution of compound 1 (14.0 g, 56.7 mmol) in THF (280 mL) wasadded BF₃-Et₂O (24.8 mL, 198.4 mmol) and ethyl 2-diazoacetate (22.7 g,199.1 mmol) at −78° C. under N₂. The reaction mixture was stirred at−78° C. for 1.5 h and then warmed to 25° C. stirred for 1.5 h. Theresulting mixture was quenched with NaHCO₃ (Sat.) and extracted with EA(600 mL). The organic layer was dried over Na₂SO₄ and concentrated invacuum to give the crude product, which was purified by flash columnchromatography to give a mixture of compound 2A and 2B (19.0 g, crude).LCMS: 334.0 [M+1].

1.1.2 Preparation of Compound 3A/3B

To a solution of compound 2A and 2B (19.0 g, crude) in MeOH (200 mL) wasadded a solution of KOH (4.8 g, 85.6 mmol) in H₂O (40 mL), and themixture was heated to 55° C. stirred for 2 h. The mixture was dilutedwith EA (800 mL) and washed with brine (600 mL). The organic layer wasdried over Na₂SO₄ and concentrated in vacuum to give the crude product,which was purified by flash column chromatography to give compound 3A(4.5 g, 31% yield) and compound 3B (3.8 g, 26% yield). Compound 3A: ¹HNMR (400 MHz, CDCl₃) δ 7.36-7.42 (m, 5H), 5.21-5.24 (m, 2H), 3.92-3.99(m, 2H), 3.54-3.57 (m, 2H), 2.41-2.45 (m, 2H), 1.58-1.73 (m, 6H).Compound 3B: ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.41 (m, 5H), 5.18 (s, 2H),3.68-3.75 (m, 2H), 3.19-3.25 (m, 2H), 2.62-2.68 (m, 2H), 2.40-2.44 (m,2H), 1.62-1.92 (m, 4H).

1.1.3 Preparation of Compound 4A

To a solution of compound 3A (1.0 g, 3.8 mmol) in EtOH (15 mL) was addedNaBH₄ (0.22 g, 5.8 mmol) at 0° C., and the mixture was stirred at 18° C.for 1 h. The resulting mixture was quenched with NH₄Cl and extractedwith EA (100 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuum to give compound 4A (0.95 g, crude), which wasused in the next step directly.

1.1.4 Preparation of Compound A02

To a solution of compound 4A (0.95 g, 3.6 mmol) in MeOH (60 mL) wasadded Pd(OH)₂/C (200 mg). The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ (15 Psi)at 25° C. for 16 hr. The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.45 g, 97%).

1.2 Preparation of Compound A03

1.2.1 Preparation of Compound 2

To a solution of CH₃M r (5.8 mL, 11.6 mmol) in THF (3 mL) was added asolution of compound 1 (1.0 g, 3.8 mmol) in THF (7 mL) at 0° C. underN₂. The reaction mixture was stirred at 20° C. for 2 h. The resultingmixture was quenched with NH₄Cl (sat.) and extracted with EtOAc (100mL). The organic layer was dried and concentrated in vacuum to give thecrude product, which was purified by flash column chromatography to givethe desired product (0.52 g, 49%).

1.2.2 Preparation of Compound A03

To a solution of compound 2 (0.52 g, 1.9 mmol) in MeOH (30 mL) was addedPd(OH)₂ (100 mg). The suspension was degassed under vacuum and purgedwith H₂ several times. The mixture was stirred under H₂ (15 Psi) at 25°C. for 16 hr. The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.26 g, 97%).

1.3 Preparation of Compound A04

1.3.1 Preparation of Compound 2

To a solution of compound 1 (0.80 g, 3.1 mmol) in EtOH (15 mL) was addedNaBH₄ (0.17 g, 4.5 mmol) at 0° C., and the mixture was stirred at 25° C.for 1 h. The resulting mixture was quenched with saturated NH₄Cl andextracted with EA (100 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuo to give compound 2 (0.76 g, crude), which was usedin the next step directly.

1.3.2 Preparation of Compound A04

To a solution of compound 2 (0.76 g, 2.9 mmol) in MeOH (40 mL) was addedPd(OH)₂/C (150 mg). The suspension was degassed under vacuum and purgedwith H₂ several times. The mixture was stirred under H₂ (15 Psi) at 25°C. for 16 hr. The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.37 g, 99%).

1.4 Preparation of Compound A05

1.4.1 Preparation of Compound 2

To a mixture of MeM r (3 M, 3.83 mL, 3.00 eq) in THF (30 mL) was addedbenzyl 4-oxoazocane-1-carboxylate (1.00 g, 3.83 mmol, 1.00 eq) in THF(30 mL) at 0° C. under N₂. The mixture was stirred at 0° C. for 10 min,then warmed to 15° C. and stirred for 2 hours. TLC showed the reactionwas completed, the mixture was poured into saturated NH₄Cl (50 mL) andstirred for 20 min. The aqueous phase was extracted with EA (40 mL*2).The combined organic phase was washed with saturated brine (20 mL),dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum, theresidue was purified by silica gel chromatography (PE/EA=10/1) to affordbenzyl 4-hydroxy-4-methyl-azocane-1-carboxylate (950.00 mg, 3.43 mmol,89.43% yield) as yellow oil.

1.4.2 Preparation of Compound A05

To a solution of benzyl 4-hydroxy-4-methyl-azocane-1-carboxylate (950.00mg, 3.43 mmol, 1.00 eq) in MeOH (30 mL) was added Pd(OH)₂ (200.00 mg,1.44 mmol, 0.42 eq) under N₂. The suspension was degassed under vacuumand purged with H₂ several times. The mixture was stirred under H₂balloon at 15° C. for 6 hours. TLC showed the starting material wasconsumed completely. The reaction mixture was filtered and the filtratewas concentrated to give 4-methylazocan-4-ol (400.00 mg, 2.79 mmol,81.42% yield) as yellow oil.

1.5 Preparation of Compound A06

1.5.1 Preparation of Compound 2

To a mixture of bromo(cyclopropyl)magnesium (0.5 M, 3.21 mL, 7.00 eq) inTHF (30 mL) was added benzyl 4-oxoazocane-1-carboxylate (800.00 mg, 3.06mmol, 1.00 eq) in THF (30 mL) at 0° C. under N₂. The mixture was stirredat 0° C. for 10 min, then warmed to 15° C. and stirred for 15 hours.LCMS showed the reaction was completed. The mixture was poured intosaturated NH₄Cl (50 mL) and stirred for 20 min, the aqueous phase wasextracted with EA (40 mL*2), the combined organic phase was washed withsaturated brine (20 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuum. The residue was purified by silica gelchromatography (PE/EA=10/1) to afford benzyl4-cyclopropyl-4-hydroxy-azocane-1-carboxylate (650.00 mg, 2.14 mmol,70.01% yield) as yellow oil. LCMS: 304.0 [M+1].

1.5.2 Preparation of Compound A06

To a solution of benzyl 4-cyclopropyl-4-hydroxy-azocane-1-carboxylate(650.00 mg, 2.14 mmol, 1.00 eq) in MeOH (30 mL) was added Pd(OH)2(100.00 mg, 722.44 umol, 0.34 eq) under N₂. The suspension was degassedunder vacuum and purged with H₂ several times. The mixture was stirredunder H₂ balloon at 15° C. for 6 hours. TLC showed the starting materialwas consumed completely. The reaction mixture was filtered and thefiltrate was concentrated to give 4-cyclopropylazocan-4-ol (350.00 mg,2.07 mmol, 96.63% yield) as yellow oil.

1.6 Preparation of Compound A07

1.6.1 Preparation of Compound 2

To a mixture of NaH (240.48 mg, 10.02 mmol, 1.50 eq) in THF (30 mL), wasadded 1-tert-butyl 5-ethyl 4-oxoazocane-1,5-dicarboxylate (2.00 g, 6.68mmol, 1.00 eq) in at 0° C. under N₂. The mixture was stirred at 0° C.for 0.5 h, then MeI (2.84 g, 20.04 mmol, 3.00 eq) was added to themixture at 0° C., and the mixture was stirred at 18° C. for 6 hours. TLCshowed the reaction was completed. The mixture was poured into saturatedNH₄Cl (150 mL) and stirred for 20 min. The aqueous phase was extractedwith EA (40 mL*2). The combined organic phase was washed with saturatedbrine (20 mL*2), dried over anhydrous Na₂SO₄, filtered and concentratedin vacuum. The residue was purified by silica gel chromatography(PE/EA=10/1) to afford 1-tert-butyl 5-ethyl5-methyl-4-oxo-azocane-1,5-dicarboxylate (2.00 g, 6.38 mmol, 95.54%yield) as yellow oil.

1.6.2 Preparation of Compound 3

To a mixture of 1-tert-butyl 5-ethyl5-methyl-4-oxo-azocane-1,5-dicarboxylate (2.28 g, 7.28 mmol, 1.00 eq) inMeOH (50 mL) and H₂O (10 mL), was added KOH (816.96 mg, 14.56 mmol, 2.00eq) in one portion at 18° C. under N₂. The mixture was heated to 60° C.for 3 hours. TLC showed the reaction was completed. The mixture wascooled to 18° C. and concentrated in reduced pressure. The aqueous phasewas extracted with EA (40 mL*2). The combined organic phase was washedwith saturated brine (20 mL*2), dried over anhydrous Na₂SO₄, filteredand concentrated in vacuum. The residue was purified by silica gelchromatography (PE/EA=10/1) to afford tert-butyl5-methyl-4-oxo-azocane-1-carboxylate (1.53 g, 6.34 mmol, 87.09% yield)as yellow oil.

1.6.3 Preparation of Compound 4

To a mixture of tert-butyl 5-methyl-4-oxo-azocane-1-carboxylate (1.53 g,6.34 mmol, 1.00 Eq) in EtOH (50 mL), was added NaBH₄ (287.81 mg, 7.61mmol, 1.20 Eq) in one portion at 18° C. under N₂. The mixture wasstirred at 18° C. for 2 hr. TLC showed the reaction was completed. Themixture was poured into water (100 mL). The aqueous phase was extractedwith EA (50 mL*3). The combined organic phase was washed with saturatedbrine (20 mL*2), dried over anhydrous Na₂SO₄, filtered and concentratedin vacuum. The residue was purified by silica gel chromatography(PE/EA=10/1, 5/1) to afford tert-butyl4-hydroxy-5-methyl-azocane-1-carboxylate (1.34 g, 5.51 mmol, 86.86%yield) as yellow oil.

1.6.4 Preparation of Compound A07

To a mixture of tert-butyl 4-hydroxy-5-methyl-azocane-1-carboxylate(300.00 mg, 1.23 mmol, 1.00 Eq) in MeOH (10 mL), was added HCl/MeOH (10mL, 4M). The mixture was stirred at 18° C. for 2 hr. TLC showed thereaction was completed. The mixture was concentrated to afford5-methylazocan-4-ol (200.00 mg, 1.11 mmol, 90.49% yield) as yellow oil.

1.7 Preparation of Compound A08

1.7.1 Preparation of Compound 2A/2B

To a solution of compound 1 (9.3 g, 37.7 mmol) in Et₂O (200 mL) wasadded ethyl 2-diazoacetate (6.0 g, 52.7 mmol) and BF₃-Et₂O (5.4 mL, 43.3mmol) at −78° C. under N₂. The reaction mixture was stirred at −78° C.for 1.5 h and then warmed to 25° C. for 16 h. The resulting mixture wasquenched with NaHCO₃ (Sat.) and extracted with EA (300 mL). The organiclayer was dried over Na₂SO₄ and concentrated in vacuum to give the crudeproduct, which was purified by flash column chromatography to givecompound 2A (4.3 g, 35%) and compound 2B (2.6 g, 21%).

1.7.2 Preparation of Compound 3A

To a solution of compound 2A (4.3 g, crude) in MeOH (40 mL) was added asolution of KOH (1.1 g, 19.6 mmol) in H₂O (8 mL), the mixture was heatedto 55° C. and stirred for 2 h. The mixture was diluted with EA (200 mL)and washed with brine (120 mL). The organic layer was dried over Na₂SO₄and concentrated in vacuum to give the crude product, which was purifiedby flash column chromatography to give compound 3A (1.5 g, 45%). ¹H NMR(400 MHz, CDCl₃) δ 7.33-7.44 (m, 5H), 5.12 (s, 2H), 3.34-3.46 (m, 4H),2.41-2.44 (m, 4H), 2.11-2.18 (m, 4H).

1.7.3 Preparation of Compound 4A

To a solution of CH₃M r (5.8 mL, 11.6 mmol) in THF (3 mL) was added asolution of compound 3B (1.0 g, 3.8 mmol) in THF (7 mL) at 0° C. underN₂. The reaction mixture was stirred at 20° C. for 2 h. The resultingmixture was quenched with NH₄Cl (sat.) and extracted with EtOAc (100mL). The organic layer was dried and concentrated in vacuum to give thecrude product, which was purified by flash column chromatography to givethe desired product (0.55 g, 51%). ¹H NMR (400 MHz, CDCl₃) δ 7.33-7.39(m, 5H), 5.16 (s, 2H), 3.33-3.51 (m, 4H), 1.58-2.07 (m, 8H), 1.23 (s,3H).

1.7.4 Preparation of Compound A08

To a solution of compound 4B (0.55 g, 2.0 mmol) in MeOH (30 mL) wasadded Pd(OH)₂/C (100 mg). The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ balloonat 25° C. for 16 hours. The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.26 g, 92%).

1.8 Preparation of Compound A09

1.8.1 Preparation of Compound 3

To a solution of Compound 2 (32 mL, 16.1 mmol) in THF (3 mL) was added asolution of compound 1 (0.60 g, 2.3 mmol) in THF (3 mL) at 0° C. underN₂. The reaction mixture was stirred at 20° C. for 16 h. The resultingmixture was quenched with saturated NH₄Cl and extracted with EtOAc (120mL). The organic layer was dried and concentrated in vacuum to give thecrude product, which was purified by flash column chromatography to givethe desired product (0.24 g, 34%). ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.39(m, 5H), 5.16-5.17 (m, 2H), 3.51-3.54 (m, 2H), 3.33-3.36 (m, 2H),1.85-1.87 (m, 2H), 1.60-1.71 (m, 4H), 0.93-0.97 (m, 1H), 0.34-0.37 (m,4H).

1.8.2 Preparation of Compound A09

To a solution of compound 3 (0.24 g, 0.8 mmol) in MeOH (15 mL) was addedPd(OH)₂/C (48 mg). The suspension was degassed under vacuum and purgedwith H₂ several times. The mixture was stirred under H₂ balloon at 25°C. for 16 h. The catalyst was filtered and the filtrate was concentratedin vacuo to give the crude product, which was used in the next stepdirectly (0.13 g, 97%).

1.9 Preparation of Compound A10/11

1.9.1 Preparation of Compound 2A/2B

To a mixture of compound 1A and 1B (2.5 g, 7.5 mmol), K₂CO₃ (2.1 g, 15.0mmol) in DMF (40 mL) was added CH₃I (1.6 g, 11.3 mmol) under N₂, and thereaction mixture was stirred at 16° C. for 16 h. The resulting mixturewas diluted with brine and extracted with EA (150 mL). The organic layerwas washed with brine, dried over Na₂SO₄ and concentrated in vacuum togive a mixture of compound 2A and 2B (2.5 g, crude), which was used inthe next step directly. LCMS: 348.1 [M+1].

1.9.2 Preparation of Compound 3A/3B

To a solution of compound 2A and 2B (2.5 g, crude) in MeOH (20 mL) wasadded a solution of KOH (0.73 g, 12.9 mmol) in H₂O (4 mL), the mixturewas heated to 70° C. and stirred for 2 h. The mixture was diluted withEA (100 mL) and washed with brine (80 mL). The organic layer was driedover Na₂SO₄ and concentrated in vacuum to give the crude product, whichwas purified by flash column chromatography to give compound 3A (0.82 g,39%) and compound 3B (0.68 g, 33%). Compound 3A: ¹H NMR (400 MHz, CDCl₃)δ 7.28-7.42 (m, 5H), 5.10-5.32 (m, 2H), 4.48-4.72 (m, 1H), 4.10-4.40 (m,1H), 3.28-3.39 (m, 1H), 2.81-2.88 (m, 2H), 1.35-1.85 (m, 5H), 0.93-1.05(m, 3H). Compound 3B: ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.40 (m, 5H),5.14-5.32 (m, 2H), 4.73 (s, 1H), 3.76-4.15 (m, 2H), 3.20-3.22 (m, 1H),2.32-2.76 (m, 3H), 1.45-2.07 (m, 4H), 0.99-1.04 (m, 3H).

1.9.3 Preparation of Compound 4A

To a solution of compound 3A (0.82 g, 3.0 mmol) in EtOH (15 mL) wasadded NaBH₄ (0.17 g, 4.5 mmol) at 0° C., and the mixture was stirred at16° C. for 1 h. The resulting mixture was quenched with NH₄Cl (Sat.) andextracted with EA (80 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuum to give compound 4A (0.79 g, crude), which wasused in the next step directly.

1.9.4 Preparation of Compound A10

To a solution of compound 4A (0.79 g, 2.9 mmol) in MeOH (40 mL) wasadded Pd(OH)₂/C (160 mg). The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred at 16° C. for 2 hunder H₂ atmosphere (15 Psi). The catalyst was filtered and the filtratewas concentrated in vacuum to give the crude product, which was used inthe next step directly (0.38 g, 94%).

1.9.5 Preparation of Compound 4B

To a solution of compound 3B (0.68 g, 2.5 mmol) in EtOH (15 mL) wasadded NaBH₄ (0.14 g, 3.7 mmol) at 0° C., and the mixture was stirred at16° C. for 1 h. The resulting mixture was quenched with NH₄Cl (Sat.) andextracted with EA (80 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuum to give compound 4B (0.64 g, crude), which wasused in the next step directly.

1.9.6 Preparation of Compound A11

To a solution of compound 4B (0.64 g, 2.3 mmol) in MeOH (30 mL) wasadded Pd(OH)₂/C (130 mg). The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred at 18° C. for 2 hunder H₂ atmosphere (15 Psi). The catalyst was filtered and the filtratewas concentrated in vacuum to give the crude product, which was used inthe next step directly (0.32 g, 95%).

1.10 Preparation of Compound A12

1.10.1 Preparation of Compound 2

To a suspension of NaH (0.27 g, 6.8 mmol) in THF (10 mL) was added asolution of compound 1 (1.5 g, 4.5 mmol) in THF (10 mL) at 0° C. underN₂, followed by a solution of Select F (1.9 g, 5.4 mmol) in DMF (10 mL)after 0.5 h. The reaction mixture was stirred at 18° C. for 2 h. Theresulting mixture was quenched with NH₄Cl (Sat.) and extracted with EA(150 mL). The organic layer was washed with brine, dried over Na₂SO₄ andconcentrated in vacuum to give the crude product, which was used in thenext step directly (1.8 g, crude).

1.10.2 Preparation of Compound 3

A mixture of compound 2 (1.8 g, crude) and KOH (0.52 g, 9.3 mmol) inMeOH/H₂O (48 mL, MeOH/H₂O=5:1) was heated to 70° C. stirred for 2 h. Themixture was diluted with EA (150 mL) and washed with brine (120 mL). Theorganic layer was dried over Na₂SO₄ and concentrated in vacuum to givethe crude product, which was purified by flash column chromatography togive compound 3 (0.91 g, 72%). ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.41 (m,5H), 5.13-5.14 (m, 2H), 4.86-4.88 (m, 1H), 3.67-3.99 (m, 2H), 3.10-3.20(m, 1H), 2.60-3.03 (m, 3H), 2.07-2.40 (m, 4H).

1.10.3 Preparation of Compound 4

To a solution of compound 3 (0.91 g, 3.3 mmol) in EtOH (15 mL) was addedNaBH₄ (0.18 g, 4.7 mmol) at 0° C., and the mixture was stirred at 18° C.for 1 h. The resulting mixture was quenched with NH₄Cl (Sat.) andextracted with EA (80 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuum to give Compound 4 (0.88 g, crude), which wasused in the next step directly.

1.10.4 Preparation of A12

To a solution of compound 4 (0.44 g, 1.5 mmol) in MeOH (25 mL) was addedPd(OH)₂/C (100 mg). The suspension was degassed under vacuum and purgedwith H₂ several times. The mixture was stirred at 18° C. for 2 h underH₂ atmosphere (15 Psi). The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.22 g, 95%).

1.11 Preparation of Compound A13

1.11.1 Preparation of Compound 2

To a suspension of NaH (0.27 g, 6.8 mmol, 60%) in THF (15 mL) was addeda solution of compound 1 (1.5 g, 4.5 mmol) in THF (15 mL) at 0° C. underN₂, followed by CH₃I (0.96 g, 6.8 mmol) after 0.5 h. The reactionmixture was stirred at 25° C. for 2 h. The resulting mixture wasquenched with NH₄Cl (Sat.) and extracted with EA (150 mL). The organiclayer was washed with brine, dried over Na₂SO₄ and concentrated invacuum to give the crude product, which was used in the next stepdirectly (1.7 g, crude).

1.11.2 Preparation of Compound 3

A mixture of compound 2 (1.7 g, crude) and KOH (0.17 g, 4.5 mmol) inMeOH/H₂O (33 mL, MeOH/H₂O=10:1) was heated to 70° C. and stirred for 2h. The mixture was diluted with EA (150 mL) and washed with brine (100mL). The organic layer was dried over Na₂SO₄ and concentrated in vacuumto give the crude product, which was purified by flash columnchromatography to give compound 3 (0.80 g, 65%). 1H NMR (400 MHz, CDCl₃)δ 7.28-7.40 (m, 5H), 5.13-5.15 (m, 2H), 3.45-3.71 (m, 2H), 3.15-3.20 (m,2H), 2.57-2.70 (m, 1H), 2.39-2.43 (m, 2H), 2.07-2.12 (m, 4H), 1.07-1.14(m, 3H).

1.11.3 Preparation of Compound 4

To a solution of compound 3 (0.80 g, 3.1 mmol) in EtOH (15 mL) was addedNaBH₄ (0.17 g, 4.5 mmol) at 0° C., and the mixture was stirred at 18° C.for 1 h. The resulting mixture was quenched with NH₄Cl (Sat.) andextracted with EA (80 mL). The organic layer was dried over Na₂SO₄ andconcentrated in vacuum to give compound 4 (0.80 g, crude), which wasused in the next step directly.

1.11.4 Preparation of Compound A13

To a solution of compound 4 (0.40 g, 1.4 mmol) in MeOH (25 mL) was addedPd(OH)₂/C (100 mg). The suspension was degassed under vacuum and purgedwith H₂ several times. The mixture was stirred at 18° C. for 2 h underH₂ atmosphere (15 Psi). The catalyst was filtered and the filtrate wasconcentrated in vacuum to give the crude product, which was used in thenext step directly (0.19 g, 94%).

1.12 Preparation of Compound A14

1.12.1 Preparation of Compound 2

To a solution of compound 1 (31.00 g, 312.72 mmol) in CH₃CN (500 mL) wasadded TEA (63.29 g, 625.44 mmol), Boc₂O (88.73 g, 406.54 mmol), DMAP(1.91 g, 15.64 mmol) in portions under N₂. The mixture was stirred at18° C. for 16 hours. TLC showed the reaction was completed. The mixturewas concentrated in reduced pressure at 35° C. The residue was purifiedby silica gel chromatography (PE/EA=30/1 to 5/1) to afford compound 2(41.90 g, 210.29 mmol, 67.25% yield) as yellow oil. LCMS: 200 [M+1].

1.12.2 Preparation of Compound 3

To a solution of t-BuOK (17.9 g, 160.60 mmol) in THF (600 mL) was addedMe₃SOCl (30.98 g, 240.90 mmol) in one portion under N₂. The mixture washeated to 100° C. and stirred for 2 hours. Then the mixture was cooledto −10° C., a solution of compound 2 (32.00 g, 160.60 mmol) in THF (600mL) was added dropwise under −10° C. and the formed mixture was stirredat −10 to 0° C. for 1 hour. TLC showed the reaction was completed. Themixture was added water (100 mL) and extracted with EA (200 mL*3). Thecombined organic phase was dried over anhydrous Na₂SO₄, and concentratedin vacuum. The residue was washed with PE/EA (50 mL, PE/EA=10/1) andfiltered to afford compound 3 (37.60 g, 80.34%) as white solid. LCMS:292 [M+1].

1.12.3 Preparation of Compound 4

To a solution of compound 3 (10 g, 0.034 mol) in DCE (800 mL) was added[Ir(COD)Cl]₂ (229 mg, 0.34 umol) in one portion under N₂. The mixturewas heated to 70° C. and stirred for 16 hours. TLC showed the reactionwas completed. The mixture was cooled to 18° C., and concentrated inreduced pressure at 40° C. The residue was washed with water (50 mL) andextracted with EA (100 mL*2). The combined organic phase was dried withanhydrous Na₂SO₄, concentrated in vacuum to afford compound 4 (6.7 g,crude) as oil. LCMS: 214 [M+1].

1.12.4 Preparation of Compound 5

To a mixture of compound 4 (12.9 g, 0.060 mol) in MeOH (100 mL) wasadded HCl/MeOH (30 mL, 4M). The mixture was stirred at 18° C. for 0.5 h.TLC showed the reaction was completed. The solution was washed with MeOH(30 mL*3) and concentrated to remove the solvent. Then THF/H₂O (200 mL)was added, followed by Na₂CO₃ (12.72 g, 0.12 mol), CbzCl (15.3 g, 0.09mol). The mixture was stirred at 18° C. for 2 hours. The mixture wasextracted with EA (100 mL*2). The combined organic phase was dried withanhydrous Na₂SO₄, concentrated in vacuum. The residue was purified bysilica gel chromatography (PE/EA=30/1 to 10/1) to afford compound 5(14.3 g, 96%) as yellow oil. LCMS: 248 [M+1].

1.12.5 Preparation of Compound 6A/6B

To a solution of compound 5 (13.10 g, 52.97 mmol) in Et₂O (1000 mL) wasadded BF₃.Et₂O (26.31 g, 185.40 mmol), N₂CHCO₂Et (21.15 g, 185.40 mmol)slowly at −30° C. under N₂. The mixture was stirred at −30° C. for 1hour. Then it was stirred at 18° for 16 hours. LCMS showed the reactionwas completed. The mixture was quenched with saturated NaHCO₃ (200 mL)and extracted with Et₂O (300 mL*2). The combined organic phase was driedover anhydrous Na₂SO₄, concentrated in vacuum. The residue was purifiedby silica gel chromatography (PE/EA=30/1 to 5/1) to afford compound 6(19.80 g, crude) as mixture. LCMS: 334 [M+1].

1.12.6 Preparation of Compound 7A/7B

To a solution of compound 6 (19.80 g, 59.39 mmol) as mixture in THF (400mL) was added NaH (2.85 g, 71.27 mmol, 60%) in portions under N₂ under−10° C., and it was stirred at −10° C. for 1 hour. Then a solution ofSelect F (25.25 g, 71.27 mmol) in DMF (50 mL) was added to the mixturein portions at −10° C. under N₂. The mixture was stirred at −10° C. for30 min. Then the mixture was warmed to 18° C. and stirred for 2.5 hours.LCMS showed the reaction was completed. The mixture was poured intosaturated NH₄Cl (50 mL) and extracted with EA (100 mL*2). The combinedorganic phase was dried over anhydrous Na₂SO₄, concentrated in vacuum.The residue was purified by silica gel chromatography (PE/EA=30/1 to10/1) to afford compound 7 (13.40 g, crude) as a mixture of 7A and 7B asyellow oil. LCMS: 352 [M+1].

1.12.7 Preparation of Compound 8A/8B

To a mixture of compound 7 (13.40 g, 38.14 mmol) in MeOH/H₂O (200 mL)was added KOH (4.28 g, 76.28 mmol) in one portion under N₂. The mixturewas stirred at 40° C. for 2 hours. LCMS showed the reaction wascompleted. The mixture was cooled to 18° C. and adjusted to pH=7 by HCl(4N) under 0° C. The mixture was extracted with EA (200 mL*2), theorganic layer was dried over anhydrous Na₂SO₄, concentrated in vacuum.The residue was purified by flash column chromatography to affordcompound 8A (430.00 mg, 4.22%) as yellow oil and a mixture of compound8A and 8B (4.5 g, mixture). LCMS: 280 [M+1].

1.12.8 Preparation of Compound 9A

To a solution of compound 8A (430.00 mg, 1.54 mmol) in EtOH (10 mL) wasadded NaBH₄ (87.39 mg, 2.31 mmol) in one portion at 18° C. under N₂. Themixture was stirred at 18° C. for 2 hours. LCMS showed the reaction wascompleted. The mixture was concentrated in reduced pressure at 35° C.The residue was poured into water (10 mL) and extracted with EA (50mL*2). The combined organic phase was dried over anhydrous Na₂SO₄,concentrated in vacuum. The residue was purified by silica gelchromatography (PE/EA=20/1 to 3/1) to afford compound 9A (370.00 mg,85.40%) as yellow oil. LCMS: 282 [M+1].

1.12.9 Preparation of Compound A14

To a solution of compound 9A (370.00 mg, 1.32 mmol) in MeOH (10 mL) wasadded Pd(OH)₂ (100.00 mg, 722.44 umol) in one portion at 18° C. underN₂. The suspension was degassed under vacuum and purged with H₂ severaltimes. The mixture was stirred under H₂ at 18° C. for 16 hours. TLCshowed the reaction was completed. The mixture was filtered andconcentrated in vacuum to compound A14 (180.00 mg, crude) as yellow oil.

1.13 Preparation of Compound A15

1.13.1 Preparation of Compound 2

To a solution of compound 1 (1.2 g, 3.6 mmol) in EtOH (15 mL) was addedaq KOH (11.6 mL, 1 M). The resulting mixture was stirred at 80° C. for 3hours. The mixture was concentrated to remove solvents. The residue wasdissolved in H₂O (10 mL), extracted with EA (20 mL×3). The organic layerwas dried over Na₂SO₄, filtered and the filtrates were concentrated todryness. The residue was purified by silica gel chromatography(PE:EA=10:1 to 5:1) to afford desired compound 2 (0.6 g, yield: 63.8%)as colorless oil.

1.13.2 Preparation of Compound 3

To a solution of compound 2 (0.6 g, 2.3 mmol) in EtOH (10 mL) was addedNaBH₄ (87 mg, 2.3 mmol). The mixture was stirred at 25 to 30° C. for 2hours. The mixture was concentrated to remove solvents and the residuewas dissolved in H₂O (10 mL), then the aqueous layer was extracted withEA (25 mL×3), the organic layer was dried over Na₂SO₄, filtered, thefiltrate was concentrated to give desired compound 3 (0.51 g, 85%) asyellow oil.

1.13.3 Preparation of Compound A15

To a solution of compound 3 (0.51 g, 1.9 mmol) in MeOH (10 mL) was addedPd/C (0.1 g) under N₂. The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ (15 Psi)at 25° C. for 16 hours. The mixture was concentrated under vacuum togive compound A15 (200 mg, 83.3%).

1.14 Preparation of Compound A16

1.14.1 Preparation of Compound 3

To a mixture of compound 1 (20 g, 0.124 mol) in DMF (300 mL) was addedNaH (12.0 g, 0.3 mol, 60%) at 0° C., then followed by compound 2 (16 g,0.124 mol). The reaction mixture was stirred at 25° C. for 16 hours. Themixture was quenched by NH₄Cl (600 mL), extracted with EA (150 mL×3).The organic layer was washed with brine (150 mL×3), dried over Na₂SO₄,filtered and concentrated to dryness. The residue was purified by silicagel chromatography (PE:EA=10:1 to 5:1) to afford desired compound 3 (1.2g, 4.6%) as brown solid. ¹H NMR (400 MHz, CDCl₃) δ 5.91-5.74 (m, 1H),5.65-5.41 (m, 1H), 4.28-4.17 (m, 2H), 4.05-3.89 (m, 2H), 3.79-3.66 (m,2H), 3.54-3.38 (m, 2H), 1.44 (s, 9H).

1.14.2 Preparation of Compound 4

To a solution of compound 3 (0.2 g, 0.93 mmol) in MeOH (10 mL) was addedPd/C (0.05 g) under N₂. The suspension was degassed under vacuum andpurged with H₂ several times. The mixture was stirred under H₂ (15 Psi)at 20° C. for 16 hours. The mixture was concentrated under vacuum togive compound 4 (200 mg, 93.3%).

1.14.3 Preparation of Compound A16

To a solution of compound 4 (0.2 g, 21.8 mmol) in dioxane (3 mL) wasadded HCl/dioxane (3 mL, 4M) at 0° C., the mixture was stirred at 0° C.for 1 hours. Then the mixture was concentrated to give compound A16 (0.1g, crude) as white solid.

1.15 Preparation of Compound A17/A18

1.15.1 Preparation of Compound 2A/2B

To a solution of compound 1 (1.0 g, 4.7 mmol) in THF (10 mL) was addedBH₃ (0.9 mL, 9 mmol). The mixture was stirred at 25 to 30° C. for 16hours. Then aqueous NaOH (0.30 g, 2 mL), H₂O₂ (0.8 g, 18 mmol) was addeddropwise. And the mixture was extracted with EA (25 mL×3), the organiclayer was dried over Na₂SO₄, filtered, the filtrate was concentrated todryness. The residue was purified with column chromatography on silicagel (PE:EA=1:1 to 1:2) to give compound 2A (400 mg) and compound 2B (150mg) as yellow oil.

1.15.2 Preparation of Compound A17

A solution of compound 2A (200 mg, 0.86 mmol) in HCl/dioxane (10.00 mL,4M) was stirred at 25° C. for 16 hr. TLC detected the reaction wascompleted. The solvent was evaporated to afford compound A17.

1.15.3 Preparation of Compound A18

A solution of compound 2B (200 mg, 0.86 mmol) in HCl/dioxane (10.00 mL,4M) was stirred at 25° C. for 16 hr. The solvent was evaporated toafford compound A18.

1.16 Preparation of Compound A19

1.16.1 Preparation of Compound 2

To a mixture of tert-butyl 6-hydroxy-1,4-oxazocane-4-carboxylate (1.40g, 6.05 mmol, 1.00 Eq) in DCM (100 mL), was added Dess-Martin (3.85 g,9.08 mmol, 1.50 Eq) in one portion at 20° C. under N₂. The mixture wasstirred at 20° C. for 12 hr. TLC showed the reaction was completed. Themixture was poured into saturated NH₄Cl (30 mL) and stirred for 20 min.The aqueous phase was extracted with DCM (20 mL*2). The combined organicphase was washed with saturated brine (20 mL*2), dried over anhydrousNa₂SO₄, filtered and concentrated in vacuum. The residue was purified bysilica gel chromatography (PE/EA=10/1) to afford tert-butyl6-oxo-1,4-oxazocane-4-carboxylate (1.10 g, 4.80 mmol, 79.30% yield) asyellow oil.

1.16.2 Preparation of Compound 3

To a mixture of MeM r (1.40 g, 11.78 mmol, 3.00 Eq) in THF (50 mL), wasadded tert-butyl 6-oxo-1,4-oxazocane-4-carboxylate (900.00 mg, 3.93mmol, 1.00 Eq) at 0° C. under N₂. The mixture was stirred at 0° C. for 1hr. Then heated to 20° C. and stirred for 2 hours. TLC showed thereaction was completed. The mixture was poured into NH₄Cl (50 mL) andstirred for 20 min. The aqueous phase was extracted with EA (40 mL*3).The combined organic phase was washed with saturated brine (20 mL*2),dried over anhydrous Na₂SO₄, filtered and concentrated in vacuum. Theresidue was purified by silica gel chromatography (PE/EA=15/1) to affordtert-butyl 6-hydroxy-6-methyl-1,4-oxazocane-4-carboxylate (750.00 mg,3.06 mmol, 77.80% yield) as yellow oil.

1.16.3 Preparation of Compound A19

To a mixture of tert-butyl6-hydroxy-6-methyl-1,4-oxazocane-4-carboxylate (1.00 g, 4.08 mmol, 1.00Eq) in DCM (8 mL), was added TFA (4 mL) at 20° C. under N₂. The mixturewas stirred at 20° C. for 2 hr. TLC showed the reaction was completed.The mixture was concentrated to afford 6-methyl-1,4-oxazocan-6-ol (1.50g, crude) as crude product.

1.17 Preparation of Compound A20

1.17.1 Preparation of Compound 2

To a solution of compound 1 (10.00 g, 46.89 mmol, 1.00 Eq) in DCM (20mL) was added HCl/MeOH (20 mL, 4 M). The mixture was stirred at 0° C.for 1 h. The mixture was concentrated to compound 2 (6.50 g, 43.44 mmol,92.65% yield) as white solid.

1.17.2 Preparation of Compound 3

To a mixture of compound 2 (3.50 g, 23.39 mmol, 1.00 Eq) in DCM (20 mL)was added TEA (5.92 g, 58.48 mmol, 2.50 Eq) and CbzCl (5.99 g, 35.09mmol) in one portion. The mixture was stirred at 25° C. for 16 h. Themixture was concentrated and the residue was purified by columnchromatography on silica gel (PE/EA=5/1 to 3/1) to afford compound 3(4.10 g, 16.58 mmol, 70.88% yield) as yellow soil.

1.17.3 Preparation of Compound 4

To a mixture of compound 3 (4.00 g, 16.12 mmol, 1.00 Eq) in DCM (40 mL)was added m-CPBA (6.96 g, 40.4 mmol, 2.50 Eq). The mixture was stirredat 25° C. for 2 h. The mixture was washed with NaHCO₃ (30 mL) and Na₂SO₃(30 mL), brine (30 mL), dried over Na₂SO₄ and concentrated to dryness.The residue was purified by column chromatography on silica gel(PE:EA=5:1 to 3:1) to give compound 4 (1.9 g, 45.2%).

1.17.4 Preparation of Compound 5

To a suspension of CuI (1.92 g, 10.06 mmol) in THF (15 mL) was added MeMr (1.2 g, 10.06 mmol) at −50° C., then it was stirred at −50° C. for 0.5h. Compound 4 (0.53 g, 2 mmol) was added into the mixture at −50° C.,the reaction was allowed to warm to 25° C. and stirred for 2 h, TLCshowed the material was consumed complete. The reaction was quenchedwith NH₄Cl (40 mL), extracted with EA (100 mL), the organic layer wasdried over anhydrous Na₂SO₄ and concentrated to dryness, the residue waspurified by column chromatography on silica gel (PE:EA=5:1 to 3:1) togive compound 5B (0.2 g).

1.17.5 Preparation of Compound A20

To a solution of compound 5 (100.00 mg, 374.11 umol, 1.00 Eq) in MeOH (5mL) was added Pd/C (0.02 g) under N₂. The suspension was degassed undervacuum and purged with H₂ several times. The mixture was stirred underH₂ (50 psi) at 25° C. for 16 hours. TLC (PE:EA=1:1) showed the startingmaterial was consumed completely. The reaction mixture was filtered andthe filter was concentrated to give compound A20 (50.00 mg, 300.39 umol,85.29% yield) as colorless oil.

1.18 Preparation of Compound A21/A22

1.18.1 Preparation of Compound 2A/2B

Compound 1 (1.10 g, 4.18 mmol) and HF/Et₃N (5.38 g, 33.4 mmol) werecharged in a 100 mL single-necked round bottom flask. The mixture wasstirred at 100° C. for 16 h under N₂. TLC showed the reaction wascomplete. Then the mixture was diluted in DCM (20 mL), washed with water(30 mL), dried over anhydrous Na₂SO₄ and concentrated to dryness. Theresidue was purified by column chromatography on silica gel (PE:EA=1:1)to give an inseparable mixture of compound 2A and 2B (300 mg, 25.33%) ascolorless oil.

1.18.2 Preparation of A21/A22

To a solution of compound 2A and 2B (250.00 mg, 882.49 umol, 1.00 Eq) inMeOH (10 mL) was added Pd/C under N₂. The suspension was degassed undervacuum and purged with H₂ several times. The mixture was stirred underH₂ (15 psi) at 25° C. for 16rs. TLC (PE:EA=1:1) showed the startingmaterial was consumed completely. The reaction mixture was filtered andthe filter was concentrated to give an inseparable mixture of A21 andA22 (100.00 mg, 670.42 umol, 75.97% yield) as yellow oil. The mixturewas used to prepare a final target directly and separated regiomers withsupercritical fluid chromatography.

1.19 Preparation of Compound A23

1.19.1 Preparation of Compound 3

To a solution of compound 1 (1.00 g, 5.71 mmol, 1.00 Eq) in DMF (20 mL)was added NaH (525.32 mg, 13.13 mmol, 60%, 2.30 Eq) at −10° C. andstirred at −10˜0° C. for 30 min, compound 2 (713.39 mg, 5.71 mmol, 1.00Eq) was added drop-wise at 0° C. over a period of 15 min under N₂. Thereaction mixture was stirred at 25° C. for 2 hours. TLC (PE/EA=3:1)showed the starting material was consumed completely. The reaction wasquenched by ice water slowly and then extracted with EA. The combinedorganic phase was washed with saturated brine, dried over anhydrousNa₂SO₄, filtered and concentrated in vacuum. The residue was purified bysilica gel chromatography (PE:EA=5:1) to give the pure compound 3(250.00 mg, 1.10 mmol, 19.26% yield) as colorless oil.

1.19.2 Preparation of Compound 4

A solution of compound 3 (250.00 mg, 1.10 mmol, 1.00 Eq) in THF (6 mL)and H₂O (3 mL) was stirred at 25° C. for 1 hr, TLC showed the reactionwas completed, the mixture was diluted with EA, washed with Na₂SO₃ andbrine, the organic phase was dried over anhydrous Na₂SO₄, filtered andconcentrated to give compound 4 (180.00 mg, 785.10 umol, 71.37% yield)as light yellow oil.

1.19.3 Preparation of Compound 5

A solution of compound 4 (180.00 mg, 785.10 umol, 1.00 Eq) in MeOH (10mL) was added NaBH₄ (89.10 mg, 2.36 mmol, 3.00 Eq) at 0° C. The mixturewas stirred for 1 hr at 27° C., TLC showed the reaction was completed,the mixture was poured into sat.NH₄Cl, extracted with EA, the organicphase was dried over anhydrous Na₂SO₄, filtered and concentrated to givecompound (140.00 mg, 605.30 umol, 77.10% yield) as colorless oil.

1.19.4 Preparation of Compound A23

To a solution of compound 5 (140.00 mg, 605.30 umol, 1.00 Eq) in dioxane(5 mL) was added HCl/dioxane (5 mL, 4M), the mixture was stirred at 27°C. for 1 hr, TLC showed the reaction was completed, the reactionsolution was concentrated to give compound A23 (80.00 mg, crude) aswhite solid.

1.20 Preparation of Compound A24

1.20.1 Preparation of Compound 2

To a solution of compound 1 (1.00 g, 4.04 mmol, 1.00 Eq) in DCM (15 mL)was added m-CPBA (2.05 g, 10.10 mmol, 2.50 Eq) at 27° C. and stirred for3 hr, TLC showed the reaction was completed, the mixture was dilutedwith EA and washed with sat.Na₂SO₃ and brine, the organic phase wasdried over anhydrous Na₂SO₄, filtered and concentrated, the residue waspurified by silica gel chromatography (PE:EA=5:1) to give compound 2(750.00 mg, 2.85 mmol, 70.51% yield) as yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 7.35-7.39 (m, 5H), 5.15-5.19 (m, 2H), 4.19-4.33 (m, 1H),4.15-4.16 (m, 1H), 3.94 (m, 3H), 3.47-3.50 (m, 1H), 3.32-3.35 (m, 3H),3.00-3.04 (m, 1H).

1.20.2 Preparation of Compound 3

To a solution of compound 2 (270.00 mg, 1.03 mmol, 1.00 Eq) in dioxane(4.1 mL) was added H₂SO₄ (1.4 mL), the mixture was stirred at 50° C. for4 hr, TLC showed the reaction was completed, the reaction solution wasdiluted with DCM and washed with sat.NaHCO₃ and brine, the organic phasewas dried over anhydrous Na₂SO₄, filtered and concentrated, the residuewas purified by silica gel chromatography (PE:EA=1:1) to give compound 3(120.00 mg, 426.59 umol, 41.42% yield) as colorless oil.

1.20.3 Preparation of Compound A24

To a solution of compound 3 (120.00 mg, 426.59 umol, 1.00 Eq) in MeOH(20 mL) was added Pd/C (20.00 mg, 426.59 umol, 1.00 Eq) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (15 psi) at 28° C. for 3 hr. TLC showedthe starting material was consumed completely. The reaction mixture wasfiltered and the filter was concentrated to give A24 (50.00 mg, 339.74umol, 79.64% yield) as colorless oil.

1.21 Preparation of Compound A25

1.21.1 Preparation of Compound 2

To a solution of compound 1 (499.05 mg, 2.34 mmol, 1.00 Eq) in THF (6mL) and H₂O (3 mL) was added NMO (630.50 mg, 5.38 mmol, 2.30 Eq) andK₂OsO₄ (86.22 mg, 234.00 umol, 0.10 Eq). The mixture was stirred at 25°C. for 2 hr, TLC showed the reaction was completed, the reactionsolution was diluted with EA, washed with sat.Na₂SO₃ and brine, theorganic phase was dried over anhydrous Na₂SO₄, filtered andconcentrated, the residue was purified by silica gel chromatography(PE:EA:DCM=1:1:1) to give compound 2 (300.00 mg, 1.21 mmol, 51.84%yield) as colorless oil.

1.21.2 Preparation of Compound A25

To a solution of compound 2 (300.00 mg, 1.21 mmol, 1.00 Eq) in EA (2 mL)was added HCl/EA (4 mL, 4M). The mixture was stirred at 25° C. for 1 hr,TLC showed the reaction was completed, the mixture was concentrated togive compound A25 (200.00 mg, crude) as white solid.

1.22 Preparation of A26

1.22.1 Preparation of Compound 2

A mixture of 1,2-bis(2-chloroethoxy)ethane (5.00 g, 26.73 mmol, 1.00 Eq)and NaI (12.02 g, 80.19 mmol, 3.00 Eq) in acetone (50 mL) was stirred at56° C. for 72 hr under N₂ atmosphere. Most solid sodium chloride wasformed. After filtration of the resulting sodium chloride, the solutionwas concentrated in vacuum. The residue was diluted with CH₂Cl₂ (200 mL)and the solution was washed with water (100 mL), dried over Na₂SO₄ andevaporated in vacuum. The residue was purified by chromatography onsilica gel (eluting with PE:EA=100:1 to 10:1) to afford the pure product1,2-bis(2-iodoethoxy)ethane (9.10 g, 24.60 mmol, 92.02% yield) as acolorless oil. ¹H NMR (400 MHz, CDCl₃) δ 3.80 (t, J=6.90 Hz, 4H), 3.70(s, 4H), 3.29 (t, J=6.78 Hz, 4H).

1.22.2 Preparation of Compound 3

To a mixture of 1,2-bis(2-iodoethoxy)ethane (2.00 g, 5.41 mmol, 1.00Eq), Na₂CO₃ (2.29 g, 21.64 mmol, 4.00 Eq) and LiClO₄ (2.30 g, 21.64mmol, 4.00 Eq) in MeCN (200 mL) was added phenylmethanamine (579.24 mg,5.41 mmol, 1.00 Eq). The mixture was stirred at 80° C. for 24 hr underN₂ protection. TLC showed the material was consumed, the mixture wasconcentrated, the residue was washed with water (60 mL), extracted withEA (50 mL*3), the combined organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated, the residue was purified bychromatography (silica gel, eluting with PE:EA=10:1 to 3:1) to affordproduct 7-benzyl-1,4,7-dioxazonane (370.00 mg, 1.67 mmol, 30.91% yield)as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.43 (m, 5H), 3.69-3.84(m, 10H), 2.86-3.00 (m, 4H).

1.22.3 Preparation of Compound A26

To a solution of 7-benzyl-1,4,7-dioxazonane (370.00 mg, 1.67 mmol, 1.00Eq) in MeOH (20 mL) was added Pd/C (50.00 mg) under N₂. The suspensionwas degassed under vacuum and purged with H₂ several times. The mixturewas stirred under H₂ (50 psi) at 28° C. for 24 hours.

TLC (Petroleum ether/Ethyl acetate=3:1) showed the starting material wasconsumed completely. The reaction mixture was filtered and the filterwas concentrated to give crude product 1,4,7-dioxazonane (170.00 mg,1.30 mmol, 77.61% yield) as light yellow oil.

1.23 Preparation of A27

1.23.1 Preparation of Compound A27

To a solution of compound 1 (2.00 g, 14.16 mmol) in THF (30 mL) wasadded LAH (1.61 g, 42.48 mmol) in portions at −10° C. under N₂. Themixture was stirred at 10° C. for 16 h. Then mixture was quenched by H₂O(1.6 mL), 15% NaOH (1.6 mL) and H₂O (3.2 mL). The mixture was dilutedwith THF (10 mL), filtrated, the filtrate was washed with aq.NH₄Cl (50mL), the aqueous layer was extracted with EA (50 mL*3). The organiclayer was added into HCl/Dioxane (10 mL, 4M). The mixture wasconcentrated in vacuum to afford compound A27 as brown solid (2.20 g,HCl salt, 94.92%).

1.24 Preparation of Compound A28

1.24.1 Preparation of Compound 2

A solution of compound 1 (1.00 g, 3.83 mmol, 1.00 Eq) in Et₂O (20 mL)was cooled to −35° C., BF₃.Et₂O (2.17 g, 15.31 mmol, 4.00 Eq) and ethyl2-diazoacetate (1.75 g, 15.31 mmol, 4.00 Eq) was added, the mixture wasstirred at −35-25° C. for 1 hr, then warmed to 25° C. and stirred for 1hr. TLC showed SM couldn't consumed completely, the mixture was dilutedwith EA and washed with NaHCO₃, the organic phase was dried andconcentrated, the residue was purified by silica gel chromatography(PE:EA=10:1) to give crude product compound 2 (180.00 mg, crude) asyellow oil.

1.24.2 Preparation of Compound 3

To a solution of compound 2 (180.00 mg, 518.13 umol, 1.00 Eq) in MeOH (2mL) and H₂O (4 mL) was added KOH (58.14 mg, 1.04 mmol, 2.00 Eq), themixture was stirred to reflux at 80° C. for 3 hr. LCMS showed thereaction was completed, the mixture was diluted with EA and washed withwater, the organic phase was dried over anhydrous Na₂SO₄, filtered andconcentrated, the residue was purified by flash column to give compound3 (60.00 mg, 217.91 umol, 42.06% yield) as colorless oil. ¹H NMR (400MHz, CDCl₃): δ 7.31-7.45 (m, 5H), 5.15 (S, 2H), 3.23-3.34 (m, 4H),2.43-2.45 (m, 4H), 2.10-2.17 (m, 2H), 1.82-1.92 (m, 4H). LCMS: 276.1[M+1], 298.1 [M+23].

1.24.3 Preparation of Compound 4

To a solution of compound 3 (60.00 mg, 217.91 umol, 1.00 Eq) in MeOH (5mL) was added NaBH₄ (24.73 mg, 653.73 umol, 3.00 Eq) at 0° C., themixture was stirred at 25° C. for 30 min, TLC showed the reaction wascompleted, the mixture was poured into sat.NH₄Cl (20 mL), extracted withEA (20 mL*3), washed with brine, the organic phase was dried overanhydrous Na₂SO₄, filtered and concentrated to give product compound 4(56.00 mg, 201.90 umol, 92.65% yield) as colorless oil.

1.24.4 Preparation of Compound A28

To a solution of compound 4 (56.00 mg, 201.90 umol, 1.00 Eq) in MeOH (10mL) was added Pd/C (10.00 mg, 201.90 umol, 1.00 Eq) under N₂. Thesuspension was degassed under vacuum and purged with H₂ several times.The mixture was stirred under H₂ (15 Psi) at 25° C. for 1 hr. TLC showedthe starting material was consumed completely. The reaction mixture wasfiltered and the filter was concentrated to give compound A28 (35.00 mg,crude) as white solid.

Part II General Procedure for Targets

To a solution of Compound 1 (0.3 mmol) in MeCN (3 mL) was added amine(0.3 mmol) and Et₃N (30 mg, 0.33 mmol) at rt, and the mixture wasstirred at rt for 2 h. The mixture was diluted with CH₂Cl₂ (20 mL) andwashed with water. The organic phase was concentrated in vacuo to givethe crude product, which was purified by prep-HPLC to give the desiredproduct.

Resolution of Chiral Compounds

Chiral resolution of selected compounds of the invention was performedaccording to the conditions listed in Table 2.

TABLE 2 Supercritical Fluid Chromatography Structure Compound IDResolution condition

2039 (2039_E1) (2039_E2) Instrument: SFC 80 Column: AD-5 μm. Mobilephase: A for CO₂ and B for MeOH (0.1% NH₃H₂O) Gradient: B 40% Flow rate:45 mL/min Back pressure: 100 bar Column temperature: 35° C. Wavelength:220 nm

2040 (2040_E1) (2040_E2) Instrument: SFC 80 Column: AD-5 μm. Mobilephase: A for CO₂ and B for EtOH (0.1% NH₃H₂O) Gradient: B 40% Flow rate:50 mL/min Back pressure: 100 bar Column temperature: 35° C. Wavelength:220 nm

2297 Instrument: SFC 80 Column: AD-5 μm. Mobile phase: A for CO₂ and Bfor EtOH (0.1% NH₃H₂O) Gradient: B 40% Flow rate: 45 mL/min Backpressure: 100 bar Column temperature: 35° C. Wavelength: 220 nm

2297_Trans1

2297_Trans2

2301 Instrument: SFC 80 Column: AD-5 μm. Mobile phase: A for CO₂ and Bfor EtOH (0.1% NH₃H₂O) Gradient: B 40% Flow rate: 45 mL/min Backpressure: 100 bar Column temperature: 35° C. Wavelength: 220 nm

2301_Trans1

2301_Trans2

2520 (2520_E1) (2520_E2) Instrument: SFC 80 Column: AD-10 μm. Mobilephase: A for CO₂ and B for MeOH (0.1% NH₃H₂O) Gradient: B 50% Flow rate:70 mL/min Back pressure: 100 bar Column temperature: 35° C. Wavelength:220 nm

Example: HBV Assembly Assay

The fluorescence quenching in vitro assembly HBV assay was developedaccording to a method described by Zlotnick and coworkers (NatureBiotechnology 2006, 24:358). The assay is based on the observation thatthe C-termini of the HBV core protein cluster together during capsidformation. This assay utilizes a mutant C150 HBV capsid protein whereall wild-type cysteines are mutated to alanines, but a C-terminalcysteine residue is preserved and is labeled with fluorescent BoDIPY-FLdye. HBV C150Bo protein is highly fluorescent, however the fluorescenceis drastically reduced during the capsid assembly process. Thus, theassay measures the ability and potency of test compounds to modulatecapsid assembly by monitoring the fluorescence of the labeled capsidC150Bo protein.

In a typical assay, the mutant HBV C150 protein (amino acids 1-150,C49A, C61A, C107A, 150C) is cloned into a T7 RNA-polymerase basedexpression vector, expressed in E. coli and purified to homogeneity as adimer. The purified HBV core protein is desalted and labeled withBODIPY-FL Dye.

In a non-limiting embodiment, the assembly assay is conducted in 96-wellplate format. The assembly reactions are carried out in 50 mM Hepesbuffer, pH 7.5 and 150 mM NaCl. The compounds are pre-incubated with theHBV CA protein for 15 min, and the assembly reactions are initiated byaddition of NaCl. The reaction is allowed to continue for 1 hour at roomtemperature.

To determine the effect on capsid assembly, each test compound isinitially screened at least 4 different concentrations in duplicates.Primary hits are compounds that show activity in the assembly assay at10 uM. Identified primary hits are confirmed in follow-up studies asdescribed elsewhere herein. Known modulators of HBV CA assembly, such asHAP-1 and BAY 41-4109, are used as control compounds in theseexperiments and exhibited EC₅₀ values consistent with the literature.EC₅₀ values for test compounds are determined via analysis of thedose-response curve.

Selected compounds of the invention were assayed in the HBV assemblyassay, as described above. The assembly assay was conducted in 96-wellplate format. The assembly reactions were carried out in 50 mM Hepesbuffer, pH 7.5 and 150 mM NaCl. The compounds were pre-incubated withthe HBV CA protein for 15 min, and the assembly reactions were initiatedby addition of NaCl. The reaction was allowed to continue for 1 hour atroom temperature. The 96-well plate assembly assay consistently had Z′factors greater than 0.7 and were robust and reproducible both fromplate-to-plate and day-to-day.

To determine the effect on capsid assembly, each test compound wasinitially screened at 5 different concentrations: about 30 μM, 10 μM, 3μM, 1 μM, and 0.3 μM in duplicates. Primary hits were compounds thatshow >50% activity in the assembly assay at about 10 μM and arepresentative group of these active compounds is shown in Table 3.

TABLE 3 HBV assembly assay (‘+’ indicates >50% activity at about 10 μM)Compound Activity 2039 + 2039_E1 + 2039_E2 + 2040 + 2040_E1 + 2040_E2 +2285_D1 + 2285_D2 + 2435 + 2436 + 2520 + 2520_E1 + 2520_E2 +

Example: Inhibition of HBV Replication Dot-Blot Assay

Compounds active in the HBV assembly assay are tested for their activityand toxicity in cellular assay. In the first anti-viral assay, theability of compounds to inhibit HBV replication in an HBV-producinghepatoma cell line using the dot-blot method is evaluated.

Briefly, confluent monolayers of HepG2-2.2.15 cells are incubated withcomplete medium containing various concentrations of a test compound.Three days later, the culture medium is replaced with fresh mediumcontaining the appropriately diluted test compound. Six days followingthe initial administration of the test compound, the cell culturesupernatant is collected, and cell lysis is performed. The samples areapplied onto Nylos membranes and DNA is immobilized to the membrane byUV cross-linking. After pre-hybridization, the HBV probe is added andthe hybridization is performed overnight. The membranes are exposed tothe Kodak films; antiviral activity is calculated from the reduction inHBV DNA levels (EC₅₀). The EC₅₀ for antiviral activity is calculatedfrom the dose response curves of active compounds. Assay performanceover time is monitored by the use of the standard positive controlcompounds ETV, BAY 41-4109, and HAP-1.

Compound cytotoxity (TC₅₀) is measured in this same HepG2-2.2.15 cellline using a CellTiter Blue-based cytotoxicity assay employed asrecommended by manufacturer (Promega). To confirm and expand theseresults, a second antiviral assay is carried out on active compoundsusing the stable HBV cell line HepG2.2.15 and measuring anti-HBV potencyby real-time PCR and cytotoxicity by CellTiter Blue. In this assay, 24hours after cell seeding, HepG2-2.2.15 cells are incubated with completemedium containing various concentrations of a test compound with BAY41-4109 and HAP-1 used as positive controls. After three days, theculture medium is replaced with fresh medium containing theappropriately diluted test compound. The cell culture is collected sixdays following the initial administration of the test compound, followedby HBV DNA extraction using QIAamp 96 DNA Blood Kit (Qiagen). Theextracted HBV DNA is diluted and analyzed by Real-Time PCR. A standardcurve is generated by plotting Ct value vs the amount of HBV plasmidstandard. Cytotoxicity is determined similarly to the above describedmethod by applying a dye uptake method (CellTiter Blue kit, Promega).

Selected compounds were tested for their activity and toxicity incellular assay. In the first anti-viral assay, the ability of compoundsto inhibit HBV replication in an HBV-producing hepatoma cell line usingthe dot-blot method was evaluated.

Confluent monolayers of HepG2-2.2.15 cells were incubated with completemedium containing various concentrations of a test compound. Three dayslater, the culture medium was replaced with fresh medium containing theappropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant wascollected, and cell lysis was performed. The samples were applied ontoNylos membranes and DNA was immobilized to the membrane by UVcross-linking. After pre-hybridization, the HBV probe was added and thehybridization was performed overnight. The membranes were exposed to theKodak films; antiviral activity was calculated from the reduction in HBVDNA levels (EC₅₀). The EC₅₀ for antiviral activity was calculated fromthe dose response curves of active compounds. Assay performance overtime was monitored by the use of the standard positive control compoundsETV, BAY 41-4109, and HAP-1. Results for selected compounds of theinvention are illustrated in Table 4.

Cytotoxity (CC₅₀) was measured in this same HepG2-2.2.15 cell line usinga CellTiter Blue-based cytotoxicity assay employed as recommended bymanufacturer (Promega).

TABLE 4 “Activity” represents activity in dot-blot-assay (‘+’indicates >50% activity at 10 μM) Compound Activity 2039 + 2039_E1 +2039_E2 + 2040 + 2040_E1 + 2040_E2 + 2285_D1 + 2285_D2 + 2435 + 2436 +2520 + 2520_E1 + 2520_E2 +

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of reducing the viral load associated with an HBV infectionin an individual in need thereof, comprising administering to theindividual a therapeutically effective amount of a compound of FormulaI:

or a pharmaceutically acceptable salt thereof; wherein R⁴ is H or C₁-C₃alkyl; R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂,—H₂PO₄, —C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆heteroalkyl, —C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, C₆-C₁₀ aryl,C₅-C₉ heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄ alkyl-(C₅-C₉heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂; R² is, independently at eachoccurrence, —OH, halo, —CN, —NO₂, R⁶, or OR⁶, wherein R⁶ is,independently at each occurrence, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl,—C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, C₆-C₁₀ aryl, C₅-C₁₀heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄ alkyl-(C₅-C₁₀heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂; Cy is

wherein R¹¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂,—C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl,—C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, C₆-C₁₀ aryl, C₅-C₉heteroaryl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), —C₁-C₄ alkyl-(C₃-C₁₀heterocycloalkyl), —C₁-C₄ alkyl-(C₆-C₁₀ aryl), or —C₁-C₄ alkyl-(C₅-C₉heteroaryl), wherein the alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl groups are optionally substituted1-5 times with halo, —OH, —CN, or —NO₂, or two R¹¹ groups, together withthe carbons to which they are attached, join to form a cyclic phosphatering; m is 0, 1, 2, 3, or 4; x is 0, 1, 2, 3, 4, or 5; and y is 0, 1, 2,3, or
 4. 2. The method of claim 1; wherein R⁴ is H; m is 0, 1, 2, or 3;x is 0, 1, 2, or 3; and y is 0, 1, 2, or
 3. 3. The method of claim 1;wherein R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂,—C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl,—C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, —C₁-C₄ alkyl-(C₃-C₁₀cycloalkyl), or —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl), wherein thealkyl group is optionally substituted 1-5 times with halo or —OH.
 4. Themethod of claim 1; wherein R² is, independently at each occurrence, —OH,halo, —CN, —NO₂, R⁶, or OR⁶, wherein R⁶ is, independently at eachoccurrence, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl,—C₃-C₁₀ heterocycloalkyl, —C₁-C₄ alkyl-(C₃-C₁₀ cycloalkyl), or —C₁-C₄alkyl-(C₃-C₁₀ heterocycloalkyl), wherein the alkyl group is optionallysubstituted 1-5 times with halo or —OH.
 5. The method of claim 1;wherein R¹¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂,—C₁-C₆ alkyl, —O—C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —O—C₁-C₆ heteroalkyl,—C₃-C₁₀ cycloalkyl, —C₃-C₁₀ heterocycloalkyl, —C₁-C₄ alkyl-(C₃-C₁₀cycloalkyl), or —C₁-C₄ alkyl-(C₃-C₁₀ heterocycloalkyl), wherein thealkyl group is optionally substituted 1-5 times with halo or —OH.
 6. Themethod of claim 1; wherein R¹¹ is, independently at each occurrence,—OH, halo, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, or—C₃-C₁₀ heterocycloalkyl.
 7. The method of claim 1; wherein R⁴ is H;each R¹ is, independently at each occurrence, —OH, halo, —CN, —NO₂, or—C₁-C₆ alkyl; R² is selected from —OH, halo, —C₁-C₆ alkyl, —C₁-C₆heteroalkyl, —C₃-C₁₀ cycloalkyl, and —C₃-C₁₀ heterocycloalkyl, whereinthe alkyl and cycloalkyl groups are optionally substituted 1-5 timeswith halo; Cy is

wherein R¹¹ is, independently at each occurrence, —OH or halo; m is 0, 1or 2; and x is 0, 1, 2, or
 3. 8. The method of claim 1; wherein R⁴ is H;each R¹ is, independently at each occurrence, —OH or halo; R² isselected from —OH, halo, and —C₁-C₆ alkyl, wherein the alkyl group isoptionally substituted 1-5 times with halo; Cy is

wherein R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₆alkyl, —C₁-C₆ heteroalkyl, —C₃-C₁₀ cycloalkyl, or —C₃-C₁₀heterocycloalkyl; m is 0, 1 or 2; and x is 0, 1, 2, or
 3. 9. The methodof claim 1; wherein R⁴ is H; each R¹ is, independently at eachoccurrence, —OH or halo; R² is selected from halo and —C₁-C₃ alkyl,wherein the alkyl group is optionally substituted 1-3 times with halo;Cy is

wherein R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃alkyl, —C₁-C₄ heteroalkyl, —C₃-C₇ cycloalkyl, or —C₃-C₇heterocycloalkyl; m is 0, 1 or 2; and x is 0, 1, 2, or
 3. 10. The methodof claim 1; wherein R⁴ is H; each R¹ is, independently at eachoccurrence, halo; R² is selected from halo and —C₁ alkyl, wherein thealkyl group is optionally substituted 1-3 times with halo; Cy is

wherein R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃alkyl, or —C₃-C₇ cycloalkyl; m is 0, 1 or 2; and x is 2 or
 3. 11. Themethod of claim 1; wherein R⁴ is H; each R¹ is, independently at eachoccurrence, halo; R² is selected from halo and —C₁ alkyl, wherein thealkyl group is optionally substituted 1-3 times with halo; Cy is

wherein R¹¹ is, independently at each occurrence, —OH, halo, —C₁-C₃alkyl, or —C₃-C₇ cycloalkyl; m is 0, 1 or 2; and x is 2 or
 3. 12. Themethod of claim 11, wherein the compound is selected from:

13-21. (canceled)
 22. The method of claim 1, further comprisingadministering to the individual at least one additional therapeuticagent selected from the group consisting of a HBV polymerase inhibitor,immunomodulatory agents, pegylated interferon, viral entry inhibitor,viral maturation inhibitor, capsid assembly modulator, reversetranscriptase inhibitor, a cyclophilin/TNF inhibitor, a TLR-agonist, andan HBV vaccine, and a combination thereof.
 23. The method of claim 22,wherein the therapeutic agent is a reverse transcriptase inhibitor, andis at least one of Zidovudine, Didanosine, Zalcitabine,2′,3′-dideoxyadenosine, Stavudine, Lamivudine, Abacavir, Emtricitabine,Entecavir, Apricitabine, Atevirapine, ribavirin, acyclovir, famciclovir,valacyclovir, ganciclovir, valganciclovir, Tenofovir, Adefovir,cidofovir, Efavirenz, Nevirapine, Delavirdine, and Etravirine. 24.(canceled)
 25. The method of claim 22, wherein the therapeutic agent isan interferon selected from the group consisting of interferon alpha(IFN-α), interferon beta (IFN-β), interferon lambda (IFN-λ), andinterferon gamma (IFN-γ).
 26. The method of claim 25, wherein theinterferon is interferon-alpha-2a, interferon-alpha-2b, orinterferon-alpha-n1.
 27. The method of claim 25, wherein theinterferon-alpha-2a or interferon-alpha-2b is pegylated.
 28. The methodof claim 26, wherein the interferon-alpha-2a is pegylatedinterferon-alpha-2a (PEGASYS). 29-31. (canceled)
 32. The method of claim1, further comprising administering to the individual at least one HBVvaccine, a nucleoside HBV inhibitor, an interferon or any combinationthereof.
 33. The method of claim 32, wherein the HBV vaccine is selectedfrom the group consisting of RECOMBIVAX HB, ENGERIX-B, ELOVAC B,GENEVAC-B, and SHANVAC B.
 34. (canceled)
 35. (canceled)